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Originally published as JCO Early Release 10.1200/JCO.2005.03.7184 on January 17 2006 © 2006 American Society of Clinical Oncology. Additional Genetic High-Risk Features Such As 11q Deletion, 17p Deletion, and V3-21 Usage Characterize Discordance of ZAP-70 and VH Mutation Status in Chronic Lymphocytic LeukemiaFrom the Department of Internal Medicine, University of Ulm; Hämatologische-onkologische Praxis Brudler, Heinrich, Bangerter, Augsburg; Central Unit Biostatistics, DKFZ; Division of Molecular Genetics, DKFZ, Heidelberg, Germany Address reprint requests to Stephan Stilgenbauer, MD, Department of Internal Medicine III, University of Ulm, Robert-Koch-Straße 8, 89081 Ulm, Germany; e-mail: stephan.stilgenbauer{at}uniklinik-ulm.de
PURPOSE: Immunoglobulin heavy chain variable-region (VH) gene mutation status and zeta-associated protein 70 (ZAP-70) expression are correlated in chronic lymphocytic leukemia (CLL), but their concordance is variable. The goal of this study was to elucidate additional factors potentially characterizing their discordance. PATIENTS AND METHODS: We evaluated ZAP-70 expression by flow cytometry, VH status by DNA sequencing, and genomic aberrations by fluorescence in situ hybridization in 148 CLL patients. The parameters were analyzed for their associations and their individual prognostic impact. RESULTS: ZAP-70 expression and VH mutation status were strongly associated in CLL without additional genetic high-risk-features as defined by the absence of 11q or 17p deletion and V3-21 usage (concordance 84%). In contrast, the proportion of discordant cases was significantly higher (39%), if such additional genetic high-risk features were present. Discordant cases with V3-21 usage were almost exclusively ZAP-70 positive and VH mutated (89%), whereas all but one of the discordant cases with high-risk aberrations were ZAP-70 negative and VH unmutated (92%). By multivariate regression analysis, two models were developed, which both include high-risk genomic aberrations and, alternatively, VH mutation status and V3-21 usage or ZAP-70 expression as independent outcome predictors. CONCLUSION: There were characteristic modes of discordance between ZAP-70 and VH mutation status depending on the presence or absence of additional genetic high-risk features such as 11q and 17p deletion or V3-21 usage. Although the biologic background for these findings is yet to be determined, these data have biologic and clinical implications regarding ZAP-70 as a pathogenic factor and outcome predictor, respectively.
The clinical course of chronic lymphocytic leukemia (CLL) is highly variable with survival times ranging from months to decades.1-3 The clinical staging systems according to Rai4 and Binet5 are of prognostic importance, but they cannot predict the outcome of individual patients in early clinical stages. To develop risk adapted treatment strategies, prognostic factors are needed, which allow the prediction of the individual patients clinical course.6 The VH mutation status and genomic aberrations have been shown to be independent prognostic factors in CLL.7-11 In addition, the rearrangement of a specific variable-region (VH) gene, the V3-21 gene, has been associated with an unfavorable clinical outcome irrespective of the VH mutation status.12-14,14A Therefore, we have considered V3-21 using cases as a separate risk group independent of the VH mutation status. Zeta-associated protein 70 (ZAP-70) is physiologically involved in the signal transduction of the T-cell receptor (TCR).15,16 Normal "B" lymphocytes do not express ZAP-70, but microarray analyses have shown that ZAP-70 is overexpressed in VH unmutated CLL and could therefore serve as a surrogate marker for the VH mutation status.17,18 In vitro studies demonstrated that ZAP-70 is involved in the signal transduction cascade initiated by B cell receptor (BCR) stimulation in VH unmutated CLL, implicating a functional role in pathogenesis and clinical behavior.19,20,3 Most importantly, a strong correlation between ZAP-70 expression and the clinical course of CLL has been reported, but ZAP-70 and VH mutation status showed a variable degree of concordance in different studies ranging from 77% to 95%.17,21-26 The causes for these interstudy differences are not clear and it is not known whether discordance of ZAP-70 and VH status may be characterized by specific patterns of additional genetic features of independent prognostic impact in CLL. To address these questions, we analyzed ZAP-70 expression, the VH mutation status, and other potentially independent biologic prognostic factors such as V3-21 usage and genomic aberrations in a large cohort of CLL patients.
Patient Cohort In the current study, 148 CLL patients observed at our institution were enrolled after informed consent. ZAP-70 flow cytometry was performed in all enrolled cases. VH sequencing and fluorescence in situ hybridization (FISH) analysis were performed in all cases where suitable samples were available (133 and 144 cases, respectively). Clinical data were available for 139 cases. The median age at diagnosis was 57 (range, 25 to 79). The male to female ratio was 1.84. At the time of diagnosis, the distribution of Binet stages A, B, and C was 65%, 27%, and 8%, respectively. The Binet stage at diagnosis was unknown in 16 cases. The median times from diagnosis to ZAP-70, VH, and FISH analyses were 28, 26, and 26 months, ranging from 0 to 260 months, respectively. At the time of ZAP-70 analysis, 72% of patients were untreated. Eighty-six patients required therapy and 23 deaths occurred within a median follow-up time of 53 months.
ZAP-70 Expression Analysis by Flow Cytometry
Analysis of Genomic Aberrations and VH Status
Statistical Analysis
Association Between ZAP-70 Expression, VH Mutation Status, and Additional Genetic High-Risk-features Such As 11q or 17p Deletion and V3-21 Usage Sixty-five of 148 (44%) cases showed ZAP-70 expression in less than 20% of cells, and were considered ZAP-70 negative, whereas 83 of 148 (56%) cases exhibited ZAP-70 expression in  20% of cells, and were considered ZAP-70 positive. VH mutation analysis was available for 133 cases. VH genes were mutated in 53 of 133 (40%) cases and unmutated in 80 of 133 (60%) cases. The V3-21 gene was clonally rearranged in 16 of 133 (12%) cases and was mutated in 11 of 16 (69%) cases. Genomic aberrations were detected in 104 of 144 (72%) cases. 13q deletion was present in 75 of 144 (52%), 13q deletion as a single aberration in 48 of 144 (33%) cases, 11q deletion in 30 of 144 (21%) cases, 17p deletion in 17 of 144 (12%) of cases, trisomy 12 in 12 of 144 (8%) cases, 14q involving aberrations in 6 of 144 (4%), and 6q deletion in 2 of 144 (1%). Concordance of ZAP-70 expression and VH mutation status was observed in 100 of 133 (75%) cases (ZAP-70 negative/VH mutated: 38 of> 53 (72%); ZAP-70 positive/VH unmutated: 62 of 80 [78%]). Discordance was observed in 33 of 133 (25%) cases (ZAP-70 negative/VH unmutated: 18 of 80 (23%); ZAP-70 positive/VH mutated: 15 of 53 [28%]). When comparing the association of ZAP-70 expression and VH mutation status in subgroups defined by the presence or absence of additional genetic high-risk features such as 17p deletion, 11q deletion, or V3-21 usage, striking differences were observed (Fig 1, Table 1, and Table 2). The association between ZAP-70 expression and VH mutation status was significantly stronger in the subgroup without additional genetic high-risk features as compared to the group with additional genetic high-risk features (concordance: 65 of 76 (86%) v 34 of 56 (61%); P = .002). Compared to the VH mutated/ZAP-70 negative subgroup high-risk genomic aberrations were significantly more frequently observed in the VH unmutated/ZAP-70 positive subgroup (24 of 27 (89%); P < .001; Table 1). Furthermore, the distribution of the two distinct discordance modes—ZAP-70 negative/VH unmutated and ZAP-70 positive/VH mutated—were strikingly different when comparing individual genetic high-risk categories (Table 1 and Table 2). Discordant cases with 11q or 17p deletion, but no V3-21 usage were almost exclusively ZAP-70 negative/VH unmutated (12 of 13 [92%], P < .001), whereas discordant cases with a V3-21 rearrangement were almost exclusively ZAP-70 positive/VH mutated (8 of 9 [89%]; P = .004). In contrast, a balanced distribution of the discordance modes was observed among cases without 11q or 17p deletion and no V3-21 usage (ZAP-70 negative/VH unmutated v ZAP-70 positive/VH mutated: 5 of 11 [45%] v 6 of 11 [55%]).
Prognostic Impact of ZAP-70 Expression and VH Status in Univariate Analyses The median OS and TFS of the ZAP-70 positive subgroup was significantly shorter compared with the ZAP-70 negative subgroup (median OS, 100 months v last observed death at 76 months; survival probability, 85%; P = .004; median TFS, 24 v 50 months; P = .005; Fig 2).
For survival analyses according to the VH status, the V3-21 exhibiting cases were considered as a mutation status independent prognostic subgroup and the cohort was therefore divided into three groups: (1) V3-21—cases with a V3-21 rearrangement irrespective of the mutation status; (2) VH unmutated—cases with an unmutated VH, but no V3-21 rearrangement; and (3) VH mutated—cases with a mutated VH, but no V3-21 rearrangement. The median OS and TFS in the groups V3-21 (n = 15) and VH unmutated (n = 70) were significantly shorter compared to the VH mutated group (n = 40; median OS: 94 v 111 months v last observed death at 15 months; survival probability 96%; P = .013; median TFS, 22 v 24 v 172 months; P < .001; Fig 2). Survival data were available for 31 of 33 cases with discordant ZAP-70 and VH mutation status (Table 2). Among the discordant cases without additional genetic high-risk features, the clinical course was as expected for the individual VH mutation status and not for the ZAP-70 expression in all cases. Among the discordant cases with presence of additional genetic high-risk features, a characteristic pattern was observed: the subgroup with V3-21 usage showed almost exclusively a high ZAP-70 expression despite a mutated VH status and an aggressive clinical course. In contrast, the subgroup with 11q or 17p deletion showed in almost all cases a low ZAP-70 expression despite an unmutated VH status and an aggressive clinical course (Table 2).
Prognostic Impact of ZAP-70 Expression, VH Mutation Status, V3-21 Usage, and High-Risk Genomic Aberrations in Multivariate Analyses
Based on the results of the Cox regression analyses, two prognostic models were developed (Fig 3). The first model was composed of five genetic subgroups, in which each patient was allocated to one category only: (1) 17p deletion, no V3-21 usage; (2) 11q deletion, no 17p deletion, and no V3-21 usage; (3) V3-21 usage; (4) VH unmutated without additional genetic high-risk features; and (5) VH mutated without additional genetic high-risk features. The median TFS for these categories was 15 months for 17p deletion, 20 months for 11q deletion, 22 months for V3-21 usage, 31 months for the VH unmutated group, and 172 months for the VH mutated group. In the second model, VH mutation status and V3-21 usage were replaced by the ZAP-70 status. The following four groups, in which each patient was allocated to one category only, were defined: (1) 17p deletion irrespective of the ZAP-70 status; (2) 11q deletion irrespective of the ZAP-70 status; (3) ZAP-70 positive, no high-risk genomic aberrations; and (4) ZAP-70 negative, no high-risk genomic aberrations. The median TFS for these categories was 15 months for 17p deletion, 20 months for 11q deletion, 31 months for the ZAP-70 positive group, and 86 months for the ZAP-70 negative group. The prognostic impact of the categories defined in the two models was retained when analyzing OS (Fig 3).
In this study, we analyzed the association and the prognostic impact of ZAP-70 expression, the VH mutation status, and additional independent genetic high-risk factors such as V3-21 usage and genomic aberrations in CLL. In agreement with previous reports, a strong association of high ZAP-70 expression and unmutated VH-genes was confirmed.17,21,23-25 However, discordance of ZAP-70 expression and VH mutation status occurred in 25% of cases. In previously published studies, the proportion of discordant cases ranged from 5% to 23%.21,23,24 These differences may be related to different flow cytometric detection strategies, (ie, the use of various antibody clones, unlabeled or directly fluorochrome labeled antibodies, and diverse gating strategies.) However, the methodology was successfully validated by additional approaches (ie, immunoblotting, cDNA microarray).21,23,24 Our data implicate that the majority of ZAP-70 and VH mutation status discordant cases may be explained by high rates of discrepancies in the presence of additional genetic high-risk features. The composition of the patient cohorts of published studies supports this concept. Crespo et al21 and Orchard et al23 who found high concordance rates (95% and 92%, respectively) analyzed cohorts with high proportions of Binet A patients (79% and 86%, respectively). Furthermore, in the study of Orchard et al,23 68% of cases were VH mutated, and the incidence of high-risk genomic aberrations was low (17p deletion, 4%; 11q deletion, 8%). In contrast, Rassenti et al,24 who found a strikingly lower concordance proportion (77%), analyzed a patient cohort frequently exhibiting biologic high-risk features (53% VH unmutated). Their cohort was derived from referral centers mainly enrolling clinically advanced patients, who were treated in approximately half of the cases. The data presented in this article indicate alternative, biologic mechanisms as a basis for the discordances. The association between ZAP-70 expression and the VH mutations status differed significantly according to presence or absence of additional genetic high-risk features. The proportion of discordant cases was particularly high in the subgroups with V3-21 usage and 17p or 11q deletion (39%). Interestingly, the mode of the discordances (ZAP-70 positive/VH mutated or ZAP-70 negative/VH unmutated) within these genetic high-risk categories showed striking differences. Cases with V3-21 usage were almost exclusively ZAP-70 positive/VH mutated (89%), whereas all but one of the discordant cases with high-risk genomic aberrations were ZAP-70 negative/VH unmutated (92%). In contrast, the association of ZAP-70 expression and the VH mutation status was stronger in cases without V3-21 usage, 17p deletion, or 11q deletion (84% concordance), and the distribution of the discordance categories was balanced (ZAP-70 positive/VH mutated v ZAP-70 negative/VH unmutated cases, 55% v 45%).
The current data provide evidence for a recently proposed theoretical model of ZAP-70 expression in the pathogenesis of CLL, in which active BCR signaling stimulates the malignant clone.3 A survival advantage for CLL cells as a result of the BCR related interaction with unknown antigens could explain the signs of antigen selection pressure, such as restricted VH-gene usage, particularly in the VH unmutated subgroup, restricted VHDJH-combinations (immunoglobulin heavy chain variable [VH], diversity [DH], and joining [JH] gene segments),14,31,32 and the occurrence of highly homologous VH-genes involving BCRs with nearly identical complementarity determining region 3 regions,12,13,33,34 a conserved V3-21 structure reflected by a remarkably narrow mutation range (mainly between 2% and 4%, compare Fig 1D), restricted JH6 usage, and a restricted usage the V To evaluate the clinical implications of additional genetic high-risk features (V3-21 usage, 11q or 17p deletion), we performed multivariate regression analyses. The VH mutation status, V3-21 usage, the presence of high-risk genomic aberrations, but not ZAP-70 expression were identified as independent prognostic factors. Based on the multivariate regression analysis, we developed a model including the VH mutation status, V3-21 usage, 17p deletion, and 11q deletion. The division in these prognostic categories allowed a good prediction of the clinical course in CLL. However, the association of ZAP-70 and the VH mutation status was strong in cases without additional genetic high-risk features, and the majority of V3-21 cases showed high ZAP-70 expression irrespective of the VH mutation status. Based on these observations, we tested a second model including ZAP-70 expression and high-risk genomic aberrations. The prediction of the clinical course was similar to the model including VH mutation status, V3-21 usage, and high-risk genomic aberrations. Therefore, a prognostic model based on ZAP-70 expression and genomic aberrations may be an alternative for the risk stratification of CLL patients, if VH-gene analysis is not available. The current data reveal for the first time a characteristic pattern of important prognostic genetic features such as 11q deletion, 17p deletion, and V3-21 usage in cases with discordant ZAP-70 and VH mutation status. ZAP-70 expression predicted an unfavorable clinical course in V3-21 using cases irrespective of the VH mutations status, but did not predict the unfavorable clinical course of the majority of cases with unmutated VH accompanied by 11q or 17p deletion. According to the data presented in this study, we propose that high-risk genomic aberrations are important independent factors in addition to the VH mutation status and ZAP-70 expression for the risk stratification in CLL. If high-risk genomic aberrations are present, it appears to be important to consider this in addition to the prognostic impact of the VH status and ZAP-70 status for the prediction of the clinical course. In the absence of high-risk genomic aberrations the VH status and ZAP-70 status may have similar prognostic impact, and might therefore be alternatively applied. This is reflected by the fact, that high-risk genomic aberrations (11q and 17p) were selected as significant independent factors in both prognostic models (based on the ZAP-70 or VH status) constructed by multivariate analysis. These observations have biologic implications with regard to the existence of different, BCR-dependent, and independent pathologic mechanisms in genetic subgroups of CLL, and prognostic importance with regard to the use of ZAP-70 as an outcome predictor. It is necessary to confirm these data in larger patient series. In addition, our data demonstrate the complexity of the assessment of the prognostic impact and interaction of multiple factors available in CLL, making further dissection of pathogenic pathways and the evaluation of combinations of prognostic factors in prospective trials mandatory.
The authors indicated no potential conflicts of interest.
We are indebted to C. Schöpflin, A. Schmid, S. Kless, A. Habermann, and E. Brückle for excellent technical assistance as well as C. Schleich and S. Groner for data management. We also thank Florence Cymbalista for open and productive discussion.
Supported by the Sander-Stiftung Grants No. 2003.086.1 and 2001.04.02, Deutsche Krebshilfe Grant No. 106116, and Deutsche José Carreras Leukämie-Stiftung e. V. Grant No. R 05/25. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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ABSTRACT
INTRODUCTION
2-14-light chain gene in V3-21 rearrangements.
