2003-07

Abstract

Background and Objectives. B-cell chronic lymphocytic leukemia (B-CLL) results from the accumulation of monoclonal CD5 + B cells. Despite its homogeneity at cellular level, B-CLL is clinically heterogeneous. Clinical studies indicate that CD38 + B-CLL are characterized by a more aggressive clinical course than are CD38 − B-CLL. On the basis of these studies and considering the established correlation between specific chromosome aberrations and the clinical course of B-CLL, it is possible that CD38 + B-CLL cases are also characterized by specific subsets of chromosomal alterations. Design and Methods. Comparative genomic hybridization (CGH) was performed on purified B-cells from peripheral blood of 52 patients with B-CLL in order to detect chromosome imbalance. The immunophenotype of the patients, including CD38 expression, was also determined by flow cytometry. The results of CGH experiments were then compared with CD38 expression. Results. We found a clear correlation between the presence of chromosomal imbalances and CD38 expression: 13/16 CD38 + cases had chromosome imbalances, most of them (12/13) correlated with a poor prognosis. Among the CD38 − B-CLL patients, only 8/36 displayed chromosome imbalances; the only three cases with loss in 13q as a single aberration, considered a good prognostic marker, were in this group. Moreover, we found that cytogenetic alterations were also more complex in the CD38 + B-CLL subset, since 9/10 with two or more aberrations were in the CD38 + group. Interpretation and Conclusions. Collectively, the data reinforce the value of CD38 as a prognostic factor and indicate that genotypic/phenotypic features distinguish B-CLL subsets. Key words: B-CLL, molecular cytogenetics, chromosome aberrations, immunophenotype, CD38. from the progressive accumulation of monoclonal CD5-positive B cells. Despite its homogeneity at cellular level, B-CLL is clinically heterogeneous since some patients survive for a long time without therapy, while others progress towards more advanced stages and die despite aggressive treatment. Design and Methods Clinical features of patients Fifty-two patients with B-CLL (32 males and 20 females) were studied. Their characteristics are described in n.a. data not available; *from diagnosis. © F e r r a t a S t o r t i F o u n d a t i o n haematologica/journal of hematology vol. 88 CGH To increase the sensitivity of the CGH, B cells were purified from PBMC by removing monocytes and CD3 + T cells by adherence to plastic surfaces and magnetic beads, respectively. DNA extraction and CGH were performed as described elsewhere. Digital image analysis Image acquisition, processing, and evaluation were performed as described elsewhere. Results Identification of two groups of B-CLL according to CD38 expression The purified malignant B-CLL cells from 52 patients were double stained with CD38 and CD19 monoclonal antibodies. Two groups of B-CLL, i.e. CD38 + and CD38 − B-CLL, were identified using the cut-off limit of 30%, already utilized in previous studies. CGH analysis Chromosome imbalances were detected by CGH. Two examples of merged CGH images and the relative mean profiles of ten metaphases with chromosomal imbalances are shown in Discussion Samples from 52 patients with typical B-CLL, diagnosed according to morphology and surface phenotype, were subjected to CGH analysis in order to obtain a comprehensive view of chromosomal gains and losses and to identify copy number aberrations specific for this pathology. To increase the sensitivity of CGH, we purified B-cells from the peripheral blood of B-CLL patients when there were less than 90% B cells. Twenty-one out of 52 (40%) patients showed chromosome imbalances; 11/21 had single imbalances, whereas the remaining 10 patients had two or more chromosome alterations. Thirty-three per cent of patients had received chemotherapy before cytogenetic analysis. The presence of patients subjected to therapy in the cohort can hardly be avoided in this kind of study. As an example, a recent study based on a large cohort of patients included a similar percentage of treated patients as did our study. Imbalances involving chromosomes 11, 12, 13 and 17 are among the most important factors in predicting survival: patients with 17p deletions are those with the worst prognosis, followed by patients with 11q deletion, those with 12q trisomy, and those with normal karyotypes, whereas patients with 13q deletions as the sole abnormality have the longest estimated survival times. This finding is possibly explained by the fact that our cohort included only patients with typical morphology/immunophenotype and that there were fewer patients with advanced stage disease. It has been demonstrated that atypical morphology and advanced stage disease correlate with loss in 17p. The B-CLL cases in this study could be subdivided into two groups according to the surface expression of CD38 by the malignant cells. This confirms previous findings from our laboratory and is also in line with data reported by others. The striking finding of this study was the clear correlation existing between the presence of chromosomal abnormalities and CD38 expression by the malignant cells. Thirteen out of 16 CD38 + patients also had chromosomal abnormalities, whereas, among the 36 CD38 − patients only 8 displayed chromosomal imbalances. These differences are highly significant (p=0.0001). Three out of the 8 CD38 − patients with chromosomal alterations had a loss in 13q as a single aberration, which generally correlates with a good prognosis, 3 patients had rare alterations, the prognostic value of which remains to be determined, while the remaining 2 patients had aberrations correlated with a poor prognosis (-11q; +12). Twelve of 13 CD38 + patients with chromosomal alterations displayed aberrations that are correlated with a poor clinical outcome (-11q; +12; -17p), whereas one patient had a gain in © F e r r a t a S t o r t i F o u n d a t i o n Chromosome aberrations and CD38 expression in B-CLL haematologica/journal of hematology vol. 88(07):july 2003 775 chromosome 3q, which is rarely found in B-CLL and is hence of undetermined prognostic value. Moreover, of the 10 patients with two or more chromosome imbalances (another marker of poor prognosis) detected in this study, 9 were within the CD38 + group. Remarkably, among the cases with the highest values of CD38 expression, 3 cases had simultaneous gain of chromosomes 12 and 18. Gain of chromosome 18 never appeared alone, but was always associated with gain of chromosome 12. Although this association has already been described in B-CLL by classical cytogenetic studies, 24,40 its significance and real frequency are not well documented. This is, in part, because most of the studies on chromosomal aberrations in large cohorts of B-CLL patients were performed with FISH using a panel of probes not including the chromosome 18 probe. Our data are in keeping with the recent observations that the unbalanced distribution of genomic aberrations in IgVH high mutation and low mutation subgroups might point toward a distinct biological background in such B-CLL subgroups and may in part, explain their different behaviors. 14 In the study by Kröber et al., 14 genomic aberrations and VH mutation status appeared to have a complementary role in estimating prognosis. Although CD38 expression has been proposed as an easily performed surrogate of VH mutational status analysis 4 its prognostic value is not completely clarified. Moreover, the relationship between CD38 expression and chromosomal aberrations has not been extensively studied. In a recent paper, Chevallier et al. Concerning the prognostic significance of CD38 expression in multivariate analysis, the authors suggested that a much larger group of patients was needed. Oscier et al. 36 showed that the mean survival of patients with loss in 17p was the shortest (47 months). In the present study the groups of patients with and without chromosomal alterations do not differ in terms of survival (not shown) probably since all but 2 patients are still alive. However, the simple patient in our cohort with loss in 17p at diagnosis was followed for only 1 year and was experiencing a poor clinical course. The different biological properties showed by B-CLL cells, including the expression of CD38, can help to explain the differences in the patients' outcomes. Recent studies, including those from our laboratory, 7,41 demonstrated that CD38 + B-CLL cells with unmutated VH/VL region genes have a viable IgM initiated signal transduction pathway. This pathway can lead to proliferation/differentiation or apoptosis depending on co-signals received by the cells in vitro. In contrast, most of the CD38 − mutated B-CLL cells do not respond to signals delivered to surface Ig. Therefore, the interaction between the cells and the environment via B-cell receptor is much less marked in CD38 − mutated cases than in CD38 + unmutated cases. These data suggest that CD38 + B-CLL cells are likely to be continuously stimulated via surface Ig. This is related to the fact that surface Ig, encoded by unmutated VH/VL genes, retain natural antibody activity and hence can react continuously with autoantigens in vivo. In the case of surface CD38-negative, mutated B-CLL cells, it is unlikely that the B-cell receptor can exert a promoting role on cell expansion since there is a not a viable IgM signal transducing pathway. Moreover, Ig encoded by mutated VH/VL genes rarely have natural antibody activity and, therefore, can rarely encounter the appropriate foreign antigen. Collectively, these considerations raise the issue of whether antigenic stimulation in B-CLL continues to exert a promoting effect on the growth of the malignant cells following transformation, and whether this is the reason for the clinical differences in B-CLL. Finally, it is unlikely that CD38 is solely a marker of cellular differentiation and clinical course. It is more probable that it also functions as a signaling molecule and, therefore may be directly involved in differences in disease severity. CD38 is known to play a role as an accessory molecule in B-cell receptor mediated signal transduction, 42,43 as well as regulating cell apoptosis in certain normal B-cell subsets. A number of conclusions can be drawn from this study. First, considering the increasing recognition of the importance of chromosome alterations in predicting the clinical outcome of B-CLL, the observation that chromosomal alterations are significantly more frequent within the CD38-positive cases lends further support to the prognostic value of the surface marker, CD38. Second, the finding that CD38 + cases can be subdivided into two groups (i.e. with and without chromosomal alterations) may lead to the delineation of further prognostic subsets of B-CLL. Third, the paucity of chromosomal alter- © F e r r a t a S t o r t i F o u n d a t i o