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Originally published as JCO Early Release 10.1200/JCO.2005.02.568 on May 2 2005 © 2005 American Society of Clinical Oncology. Evidence for Distinct Pathomechanisms in Genetic Subgroups of Chronic Lymphocytic Leukemia Revealed by Quantitative Expression Analysis of Cell Cycle, Activation, and Apoptosis-Associated GenesFrom the Department of Internal Medicine III, University of Ulm, Ulm; Department of Molecular Genetics and Department of Biostatistics, German Cancer Research Center, Heidelberg, Germany; Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria Address reprint requests to Stephan Stilgenbauer, Internal Medicine III, University of Ulm, Robert-Koch-Straße 8, 89081 Ulm, Germany; e-mail: stephan.stilgenbauer{at}medizin.uni-ulm.de
PURPOSE: In patients with chronic lymphocytic leukemia (CLL), the VH mutation status and genomic aberrations (13q–, +12q, 11q–, 17p–) identify distinct prognostic subgroups. The aim was to elucidate biologic mechanisms through which these genetic markers may exert their pathogenic influence. PATIENTS AND METHODS: Twenty-four genes involved in apoptosis, cell cycle, B-cell activation, and B-cell receptor (BCR) signaling were analyzed by real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) in 82 CLL cases constituting prototypic genetic CLL subgroups as defined by the VH mutation status and the genomic aberrations 13q–, +12, 11q–, and 17p–. RESULTS: The VH mutation subgroups were characterized by a differential expression of the BCR associated genes ZAP70 and PI3K. Among the subgroups defined by genomic aberrations, there was a deregulation of candidate genes from the affected critical genomic regions such as CDK4 (up), ATM (down), and TP53 (down) in the groups +12, 11q–, and 17p–, respectively. Additionally, the genomic subgroups were characterized by a significant deregulation of cell cycle and apoptosis regulators: AKT (up) in 13q, E2F1 (up) in +12, MYC (up) and BCL-2 (down) in 17p–, and CCND3 (down) in 11q– as well as 17p–. The 17p– subgroup showed an additional down-regulation of BCR-associated genes such as SYK and PI3K. CONCLUSION: The characteristic gene expression patterns observed implicate a differential regulation of cell cycle, apoptosis, and BCR signaling in the genetic subgroups illustrating distinct pathomechanisms and are evidence for a gene dosage effect being operative in CLL. These findings link the biologic diversity and clinical heterogeneity of CLL.
Chronic lymphocytic leukemia (CLL) is characterized by a variable clinical course, with survival times ranging from months to decades.1,2 This clinical heterogeneity has been shown to be related to inherent genetic features of the CLL cells such as the VH mutation status and genomic aberrations. While CLL with mutated VH displays a good prognosis, VH unmutated CLL is characterized by a poor outcome,3,4 suggesting that the different maturation stages according to the VH mutation status may define distinct CLL pathomechanisms.5 However, by genome-wide gene expression analysis using microarrays, the transcriptional profile of the VH subgroups could not be distinguished in unsupervised analyses, and only a small number of genes were found to be differentially expressed when comparing the VH subgroups by supervised analyses.6,7 Among these, ZAP70 had a prominent role, and further studies showed that B-cell receptor (BCR) crosslinking on ZAP70-positive CLL cells led to increased tyrosine phosphorylation (eg, of p72 [Syk]), indicating an increased activation after BCR stimulation as a pathomechanism.8 Additionally, genomic aberrations, which have an independent prognostic impact and may be transforming events in CLL, were not included in these studies. The most common genomic aberrations are losses at the chromosomal bands 13q14 (13q–: 55%), 11q22 to q23 (11q–: 18%), 17p13 (17p–: 7%), and gains at chromosome 12 (+12: 16%).9 17p– and 11q– identify poor prognostic subgroups in multivariate analysis.9-12 Recurrent genomic aberrations point to the loci of pathogenic genes such as ATM at 11q22-23 and TP53 at 17p13. However, the expression pattern of these and other candidate genes is so far unresolved in the subgroups of CLL defined by genomic aberrations. The aim of the present study was to elucidate pathogenic events in pathways of oncogenic potential in the genetic subgroups of CLL. To investigate candidate gene expression of genetically distinct CLL subtypes, we built prototypic subgroups composed of cases defined by the VH mutation status and genomic aberrations, ie, VH mutated (VH MUT), VH unmutated (VH UM), normal karyotype by FISH (normal), 13q– as sole abnormality (13q–), 11q–, +12, and 17p–. To obtain a highly sensitive quantitation of gene expression, we used real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) and focused on a selected set of functionally relevant genes playing central roles in the regulation of apoptosis, cell cycle, B-cell activation, and BCR signaling, namely AICL (CLECSF2), AID (AICDA), AKT1, ATM, BAX, BCL2, CCND1, CCND2, CCND3, CDK4, MYC, E2F1, P21 (CDKN1A), P27 (CDKN1B), PI3K (PIK3CB), MDM2, MCL1, NFKB1, Survivin (BIRC5), SYK, TCL (TCL1A), TP53, TRADD, and ZAP70.
Patients Peripheral blood was obtained for diagnostic procedures from all cases after informed consent and the studies were institutional review board–approved. Samples from 82 patients with CLL diagnosed according to established morphologic and immunophenotypic criteria were included. Staging information was complete for 76 patients; 30 patients were Binet A; 26, Binet B; and 20, Binet C. Median age was 57 years (range, 38 to 84 years), and 56% were male. All samples were from the same date as the corresponding VH and fluorescent in situ hybridization (FISH) analyses. Sixty-seven samples were collected at time of diagnosis (15 during the course of the disease), and all except seven patients were untreated. In these cases, mostly patients with refractory disease, one to three previous therapies had been administered, and the samples were collected in active recurrent disease after at least 2 treatment-free months. Clinical follow-up for survival analysis was available for 60 patients. Translocation t(11;14)–positive cases were not included.
Genetic Analysis
Genetic Subgroups
Sample and RNA Preparation The procedures were done as previously reported,14 including a DNAse I digestion of total RNA to avoid contamination with genomic DNA.
RQ-PCR
Data Normalization Eight serial dilutions of cDNA from tumor cell lines were used to obtain a calibration graph, which was measured in every single RQ-PCR run. In general, the B-cell CLL cell line EHEB was used for calibration. The T-cell line Jurkat and the Burkitt cell line Namalva were used for calibration of ZAP70 and AID expression levels, respectively, due to absent expression in EHEB. The average value of three independent endogenous control amplicons (PGK1, LMNB1, ACTB) were used for normalization.17
Control Experiments
Statistical Analysis
Quantitative expression levels of 24 genes (listed in Table 2 with their chromosomal position) with central roles in the regulation of apoptosis, cell cycle, BCR signaling, and B-cell activation were analyzed by RQ-PCR in 82 cases constituting the prototypic genetic CLL subgroups (VH MUT, VH UM, 13q–, normal, +12, 11q–, and 17p–). A survival probability analysis was performed to test whether the selected cases were a representative collection with regard to the clinical impact of the genetic markers to be analyzed (Fig 2). The individual subgroups VH MUT, VH UM, +12, 11q–, and 17p– showed the expected survival probabilities, demonstrating that the selected cases represent a valid collection for this study.
VH Mutation Subgroups The VH MUT versus VH UM analysis was restricted to the set of normal and 13q– cases to avoid interference of potentially distinct expression signatures of +12, 11q–, and 17p– cases. Only two of the 24 genes analyzed showed significant differential distributions of expression levels between the VH mutation subgroups (summarized in Table 3): ZAP70 levels were 3.24-fold higher in VH UM than in VH MUT cases (Fig 3A), whereas PI3K showed lower expression levels in VH UM cases (0.68-fold). CD3 RNA expression levels, used as a calibrator for T-cell contamination, were comparable between the VH subgroups, and a correction for CD3 expression did not change the results for ZAP70 significantly (data not shown). The CCND1/CCND3 ratio was higher in VH MUT. When comparing VH mutated and VH unmutated cases in the overall cohort (n = 82; Table 4) including +12, 11q–, and 17p– cases, similar results were obtained for ZAP70 and PI3K, but in addition, a down-regulation of ATM and CCND1 was evident, probably as a consequence of the inclusion of 11q– and 17p– cases, respectively (see Results section Genomic Aberrations).
Due to the potential pathogenic impact of ZAP70 expression in CLL, an additional comparison of ZAP70-high and ZAP70-low expressing cases was carried out. Therefore, 21 cases from the quartile with the highest ZAP70 expression were compared with 21 cases from the quartile with the lowest expression. In the ZAP70-high group, an upregulation of SYK (P = .02, 1.59-fold) and a down-regulation of PI3K (P = .04, 0.71-fold) were identified.
Genomic Aberrations 13q– (single) was compared with normal cases (Table 5). The most characteristic finding was a low-level but highly differential overexpression of AKT (P = .001) in 13q– cases (Fig 3B). To explore whether the differential expression of AKT was restricted to cases with a 13q– single aberration (ie, no additional abnormality) or a general phenomenon of 13q deletion, we compared all cases with a 13q deletion (n = 42) against all cases without a 13q deletion (n = 40), irrespective of additional aberrations. The higher expression of AKT was also observed when comparing the 13q deletion (irrespective of additional aberrations) with the non-13q deletion subgroup (Fig 4).
The subgroups +12, 11q–, and 17p– were individually compared with the cohort of normal and 13q- cases (n = 34, Fig 1). This comparison was chosen due to the occurrence of an additional 13q– in approximately half of the cases with +12, 11q–, and 17p–. In addition, to investigate expression characteristics without interferences of VH mutation status, a restricted analysis was carried out including only VH UM cases. In this comparison, the homogeneously unmutated subgroups +12(UM), 11q–(UM), and 17p–(UM) were compared individually with the VH UM subgroup without these aberrations.
When comparing the +12 subgroup with the normal/13q– cohort (Table 6
In the 11q– subgroup (Table 8), the most differentially expressed genes were ATM (Fig 3C) and CCND3, both with a lower expression in 11q– as compared with 13q–/normal (P < .001, 0.43-fold and P = .015, 0.66-fold, respectively). When the comparison was restricted to 11q–(UM) versus VH UM (Table 9), the lower ATM expression remained the most prominent finding (P < .001), but additional trends for upregulation of MCL and NFKB and down-regulation of SYK were noted.
The 17p– subgroup displayed the highest number of differentially expressed genes affecting all investigated pathways (Tables 10 and 11). In comparison to normal/13q–, the 17p– cases showed a significantly lower expression of CCND3 (P < .001), BCL2 (P = .001), SYK (P = .004), TP53 (P = .007; Fig 3D), ATM (P = .005), TCL (P = .006), PI3K (P = .021), CCND1 (P = .030), and AID (0.013) with a range of underexpression between 0.31- and 0.66-fold. Significant overexpression was observed for P21 (1.94-fold), MYC (2.18-fold), and AICL (3.57-fold). BCL2/BAX ratios were lower in the 17p– subgroup. The comparison of 17p–(UM) versus VH UM produced similar results. In this analysis, TP53 was the most significantly down-regulated gene (P = .003).
Figure 5 gives a comparison of median expression levels of selected candidate genes involved in apoptosis, cell cycle regulation and BCR signaling in the genetic subgroups VH MUT, VH UM, +12, 11q–, and 17p–. ZAP70 expression levels were highest in VH UM, but lower in +12, 11q–, and 17p– cases, though these were predominantly VH unmutated. Similarly, SYK expression was lower in +12, 11q–, and 17p– as compared with VH UM. While ATM expression was lowest in 11q– cases, TP53 levels were lowest in 17p– cases. Both subgroups harbor a genomic loss of the respective gene locus. Surprisingly, BCL2 showed the highest expression in the VH MUT subgroup but the lowest expression in the poor-risk 17p– subgroup. BAX expression was homogeneous except for an overexpression in +12. Whereas CCND3 expression was characteristically low in 11q– and 17p–, MYC was overexpressed in 17p–.
Candidate genes that discriminated the genomic subgroups +12, 11q–, and 17p– from normal/13q– (P  .05) were used for an outline of the major pathways in which the investigated genes are involved and the mode of their deregulation in the genetic CLL subgroups (Fig. 6A to C).
Gene Expression Inter-Relations For a general comparison of subgroup-specific expression inter-relations, all genes were clustered hierarchically by their strength of correlation (Figs 7A to E), starting with the pair of genes with the largest positive coefficient of pairwise correlation (see Patients and Methods). Using this algorithm, highly distinct, subgroup-specific expression inter-relations can be illustrated. Whereas in VH MUT the strongest cluster included positive correlations between MCL, NFKB, and P27, the cluster consisted of CCND2, TCL, and BCL2 in VH UM (Figs 7A and B). Although the subgroups +12, 11q–, and 17p– were predominantly composed of VH unmutated cases, their clustering was distinct for each of the subgroups and differed from the VH UM group. The strongest cluster in +12 consisted of PI3K, ATM, and TCL, in contrast to MCL, P21, and MDM2 in 11q–, and BAX and TRADD in 17p (Fig 7C–E).
Control Experiments To ensure the reproducibility of the results, experiments were repeated in replicate with independent measurements of six candidate genes and all housekeeping genes. Highly comparable results were obtained. Distributions with initial P values less than .05 could be reproduced in all cases, and no additional significant results were obtained (data not shown).
Although genetic factors have a strong impact on the clinical course of CLL, little is known about the pathomechanisms of the genetic subgroups. Despite their highly distinct clinical behavior it was not possible to separate the CLL subgroups with mutated and unmutated VH genes by unsupervised cluster analysis of global gene expression data derived from microarrays.6,7 However, in these studies, genomic aberrations were not included, and the analyses were not performed by a highly quantitative method such as RQ-PCR, possibly obscuring subtle expression differences in a wealth of data points. In contrast to previous expression analyses, we used unmanipulated cell samples (ie, no CD19+ selection), but selected for a high content of genetically defined tumor cells. We thereby gained a "genetic" tumor purity of approximately 85% as compared with greater than 95% "B-cell purity" by CD19 selection. A disadvantage of this approach was a potentially remaining influence of non-CLL cells in the sample. This applies particularly for ZAP70, which is strongly expressed in T-cells. Since the current study focused on subgroup comparisons, this problem may have been reduced by the balanced content of genetically defined CLL cells and contaminating T-cells (monitored by CD3) in the different subgroups. In line with pivotal studies6,7 only few differences in gene expression were observed between the VH mutation subgroups. ZAP70 was previously shown to be related to increased BCR signaling in CLL.8 In line with this, we identified elevated levels of SYK, a ZAP70-related kinase, in cases with high ZAP70 expression, arguing for a synergistic role of ZAP70 and SYK in initial BCR signaling. In contrast, PI3K expression, a gene known to be involved in BCR signaling by linking initial receptor signals from tyrosine kinases to downstream effectors,20-23 was lower in VH UM CLL. Similarly, PI3K was identified between the ZAP70 subgroups with a lower expression in ZAP70 high expressing cases. In contrast to Chen et al,8 these findings suggest an inactivity or suppression of physiologic downstream BCR cascades as a consequence of ZAP70 overexpression, potentially favoring atypical cellular responses or alternative pathways following BCR stimulation. In CLL characterized by genomic aberrations, multiple gene deregulations affecting different pathways were observed, suggesting a combined model of cell-cycle deregulation, impairment of apoptosis, and dysfunctional cellular responsiveness for these subgroups. Importantly, most deregulations between genomic subgroups were reidentified when comparing the genomic subgroups in purely VH unmutated cases, clearly demonstrating the additional impact of genomic aberrations on the gene expression pattern independently of the VH mutation status. Although 13q deletion is the most common genomic abnormality in CLL, its biologic role is still largely unresolved.24,25 In 13q–, the most characteristic finding was a low-level overexpression of AKT, which was also observed in the overall cohort of cases with 13q deletion, despite the inclusion of heterogeneous cases with secondary genetic events (11q–, +12, 17p–). Therefore, AKT overexpression seems to be a general phenomenon of 13q deletion potentially providing a new pathomechanism for this genetic abnormality, and not a phenomenon linked only to the favorable 13q– (single) group. AKT can be activated by the BCR and promotes B-cell survival by inactivating components of the apoptotic machinery.26,27 In CLL cells, CD40-crosslinking induced cell proliferation, which was associated with an increase of AKT.28 Additionally, AKT activation was shown to protect CLL cells from chlorambucil- and radiation-induced apoptosis.29 This might explain a survival advantage for tumor clones with enhanced AKT expression, thereby favoring cells with a 13q deletion. Whether AKT overexpression could result from a deregulation of candidate genes from the critical genomic region on 13q14, has to be assessed. Remarkably, in +12, all of the differentially expressed genes (E2F1, BAX, P27, CDK4) were overexpressed. Overexpression of CDK4 and P27 might be explained by a direct gene dosage-dependant upregulation due to their location on chromosome 12. The transcription factor E2F1 is a critical determinant of the G1/S-phase transition during cell cycle, leading to the transcription of S-phase proteins.30-32 Overexpression of E2F1 could be correlated with proliferation in human cancers and B-cell lymphomas.33,34 Therefore, E2F1 overexpression in +12 suggests enhanced proliferative activity as a potential pathomechanism in the evolution of this genetic abnormality. In addition, E2F1 activity is known to be regulated through the action of G1 cyclins and associated kinases such as CDK4.30-32 Therefore, overexpression of CDK4 might pathomechanistically contribute to E2F1 expression in +12. Deletion in chromosome bands 11q22-23 is associated with extensive lymph node involvement and poor survival in CLL.9 ATM is located within the minimally deleted region and coordinates responses to DNA damage and is able to induce repair mechanisms and apoptosis by activating the TP53 pathway,35 suggesting that ATM might be acting as a tumor suppressor gene. However, only about one third of the CLL cases with 11q– exhibit ATM mutations of the second allele,36,37 implicating the requirement of additional ATM inactivation mechanisms in CLL with 11q–. In this study, we demonstrate that the majority of 11q– cases (13 of 16) exhibit reduced ATM transcript levels, pointing to a gene dosage-dependant down-regulation. Pettitt et al showed that p53 dysfunction in CLL may occur independently of TP53 mutations.38 Interestingly, these cases were associated with ATM mutations and exhibited reduced ATM protein expression. Therefore, down-regulation of ATM transcription in 11q- CLL may be a pathogenic factor contributing to dysfunctional apoptosis and dismal clinical outcome. The largest number of deregulated genes was detected in the 17p- subgroup possibly reflecting the distinctly aggressive biology of this subgroup. This finding is in contrast to Stankovic et al39 reporting an indistinguishable native gene expression pattern when comparing wild-type and TP53-mutant CLL. The limited number of cases studied by Stankovic et al and the higher sensitivity of RQ-PCR in comparison to the microarray approach may account for this discrepancy. The characteristic two-fold reduction of TP53 transcript observed in our series indicates a gene-dosage effect as additional or synergistic mechanism contributing to TP53 dysfunction in CLL. Of note, we detected significantly reduced ATM levels in cases with 17p–, a finding that might contribute synergistically to apoptosis impairment in the 17p– subgroup. The consensual down-regulation of ATM in the 11q– and 17p– subgroup argues for related pathogenic pathways involving a common mechanism of ATM inactivation as previously suggested.38,39 Overexpression of P21 despite low TP53 expression in 17p– appears unexpected and may indicate TP53-independent counter-regulation of P21 in 17p– CLL. The finding of particularly low BCL2 levels in 17p–, the subgroup with the worst clinical course, was surprising due to the known antiapoptotic function of BCL2 and the association of high BCL2/BAX ratios with aggressive disease.40-42 This finding argues against a relevant role of BCL2 overexpression or elevated BCL2/BAX ratios in the pathogenesis of the genetic CLL subgroups or in CLL prognosis. In addition to the down-regulation of apoptosis-associated genes in 17p–, there was a profound deregulation of cell-cycle regulators, including CCND3, CCND1, and MYC. CCND1 and, more pronounced, CCND3, showed reduced transcript levels in 17p–. However, as a consequence of this consensual down-regulation, the relationship between the transcript levels of both genes (CCND1/CCND3 ratio) remained largely unchanged. In contrast to the cyclins, MYC showed a differential overexpression in 17p–. MYC is known as a potent activator of cell proliferation,43 and MYC expression has been suggested as a marker for poor prognosis in B-cell lymphomas,44-46 implicating enhanced proliferative activity as an additional pathomechanism of the 17p– subgroup. A common phenomenon observed in the subgroups +12, 11q–, and 17p– was the deregulation of genes from critical genomic regions implicating a gene-dosage effect being operative in CLL, which is in line with a recent report.47 In addition to the differential expression of individual genes described in this work, cluster analysis of gene expression correlations lead to subgroup-specific patterns reinforcing the concept of pathomechanisms distinct for the genetic CLL subgroups and thereby linking the biologic diversity and clinical heterogeneity of CLL.
The authors indicated no potential conflicts of interest.
Supported by the DFG (Sti 296/1-1), Deutsche Krebshilfe (70-3183-Li1), Wilhelm Sander Stiftung (2002.095.1), Jubiläumsfonds of the Austrian National Bank (No. 9964; U.J.). Presented at the Annual Meeting of the American Society of Hematology, San Diego, CA, 2003. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
statistic and plotted as a red and blue color image of the correlation matrix. Increasingly positive correlations are represented with reds of increasing intensity, and increasingly negative correlations are represented with blues of increasing intensity. To present correlation matrices we started with the pair of genes with the largest positive coefficient of pairwise correlation. The next gene selected is the one whose expression values show the strongest positive correlation with the mean expression of the two previously selected genes. This procedure is continued by selecting the gene whose expression values have the strongest positive correlation with the mean expression of all the genes previously selected. 
