Spotlight on Chronic Lymphocytic Leukemia

Genetics of chronic lymphocytic leukemia: genomic aberrations and VH gene mutation status in pathogenesis and clinical course

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Abstract

The genetic characterization of chronic lymphocytic leukemia (CLL) has made significant progress over the past few years. While conventional cytogenetic analyses only detected chromosome aberrations in 40–50% of cases, new molecular cytogenetic methods, such as fluorescence in situ hybridization (FISH), have greatly enhanced our ability to detect chromosomal abnormalities in CLL. Today, genomic aberrations are detected in over 80% of CLL cases. Genes potentially involved in the pathogenesis were identified with ATMin a subset of cases with 11q deletion and p53 in cases with 17p13 deletion. For the most frequent aberration, the deletion 13q14, candidate genes have been isolated. Genetic subgroups with distinct clinical features have been identified. 11q deletion is associated with marked lymphadenopathy and rapid disease progression. 17p deletion predicts for treatment failure with alkylating agents, as well as fludarabine and short survival times. In multivariate analysis 11q and 17p deletions provided independent prognostic information. Recently, another important issue of genetic risk classification in CLL was identified with the mutation status of the immunoglobulin variable heavy chain genes (VH). CLL cases with unmutated VH show more rapid disease progression and shorter survival times. Whether CD38 expression can serve as a surrogate marker for VH mutation status is currently discussed controversially. VH mutation status and genomic abnormalities, such as 17p and 11q deletion, have recently been shown to be related to each other, but were of independent prognostic information in multivariate analysis. Moreover, genomic aberrations and VH mutation status appear to give prognostic information irrespective of the clinical stage and may therefore allow a risk assessment for individual patients early in the course of their disease.

Introduction

In a review by Mitelman and Levan on chromosome aberrations in human neoplasia no specific aberration was associated with chronic lymphocytic leukemia (CLL) in 1978.1 Since then our knowledge of cytogenetic and molecular cytogenetic findings in this disease has increased tremendously. In the late 1970s, specific chromosomal aberrations in CLL were identified through the use of B cell mitogens. Clonal chromosome abnormalities are detected in 40–50% of cases by conventional cytogenetics.2,3 Chromosome banding studies are still hampered by the problem of the low in vitro mitotic activity of the CLL cells. In recent years, modern molecular cytogenetic methods, such as fluorescence in situ hybridization (FISH), made it possible to identify chromosome aberrations in approximately 80% of CLL cases using a disease-specific probe set.4 Deletion 11q22-q23 and deletion 17p13 are independent prognostic markers in multivariate analysis identifying subgroups of patients with rapid disease progression and short survival times. On the other hand, deletion of chromosome band 13q14 as the sole abnormality is associated with a favorable prognosis.4 With the exception of p53 and ATM, most of the affected tumor suppressor genes and oncogenes in CLL are as yet unknown.

Another important genetic marker is related to the stage of differentiation of the CLL cells. The recombination of variable (V), diversity (D) and joining (J) immunoglobulin gene segments and the insertion of nontemplated nucleotides at the V–D and D–J junction occur physiologically in the pre-germinal center phase of B cell differentiation. In the following germinal center phase, the variable region genes (VH) of the B cells can be modified by somatic hypermutation through introduction of point mutations and occasional deletions and duplications at a very high rate.5 Initially, it was thought that the clonal accumulation of CD5+ B cells in CLL was comprised of antigen-inexperienced lymphocytes with VH in germline configuration corresponding to naive B cells. However, recent studies showed the presence of significant somatic mutation of the VH genes, indicating that approximately in half of all CLL cases the neoplastic cells correspond to postgerminal-center memory B cells.6,7 In pivotal studies Damle et al8 and Hamblin et al9 have shown that the presence of unmutated VH genes predicts for an inferior survival in CLL.

Characterization of genomic aberrations and the VH mutational status may help to understand the pathogenesis of CLL and may give prognostic information independent from conventional clinical markers for a risk-adapted management of CLL patients.

Methodological approaches for the genetic analysis of CLL

Conventional chromosome banding analysis

With the aid of B cell mitogens such as TPA, Epstein–Barr virus, lipopolysaccharide, pokeweed mitogen, cytochalasin B, anti-human IgM, B cell growth factor, and an anti-CD40 antibody recurrent chromosome aberrations were identified in the 1980s.10,11,12,13,14,15,16,17 Despite the use of these mitogens, conventional chromosome banding analysis has remained difficult in CLL and even with improved culture techniques clonal genomic abnormalities can be detected in only 40–50% of CLL cases.2,3 In cases with normal karyotype a study combining immunophenotyping and karyotype analysis showed that metaphase spreads without clonal aberrations often originate from non-leukemic T-lymphocytes.18

Comparative genomic hybridization

Without knowledge of candidate regions involved in a specific tumor type, a genome-wide screening for chromosome imbalances can also be performed by comparative genomic hybridization (CGH).19,20,21 As this method works with differently labeled total genomic DNA samples (normal tissue DNA vs tumor DNA) that are co-hybridized to normal metaphase spreads, no tumor cell metaphase spreads are needed. In CLL, comparison of CGH data with banding results showed that the incidence of aberrations detected by CGH was higher.22

Fluorescence in situ hybridization (FISH)

The development of fluorescence in situ hybridization (FISH) in the late 1980s and early 1990s, provided a very powerful tool for the detection of chromosome aberrations in tumors,23 especially in CLL. With the aid of specific DNA probes genomic abnormalities can be detected by FISH on the single cell level in interphase nuclei or metaphase spreads. Therefore, this approach is also referred to as ‘interphase cytogenetics’ (see also Figure 1).24 At the molecular level, many critical genomic regions have only very recently been characterized in CLL and in contrast to PCR-based methods no sequence information of the region under investigation is required. Another major advantage of FISH compared to conventional banding analysis or CGH is a higher spatial resolution for the detection of genomic aberrations resulting in a higher sensitivity. Therefore, the different findings regarding the spectrum and frequency of genomic abnormalities seen in various FISH studies compared to banding studies are not surprising.25 Using the molecular cytogenetic FISH approach with a comprehensive probe set, today genomic aberrations are detected in approximately 80% of CLL cases.4

Figure 1
figure1

Examples of FISH images demonstrating genomic aberrations in CLL. (a) CLL with monoallelic 17p13 deletion as demonstrated by the single red signal in five of the six nuclei shown. Two green signals of an internal control probe hybridizing to an adjacent disomic genomic region proof a high hybridization efficiency. The single cell with two red signals likely represents a non-leukemic cell from the blood specimen. (b) CLL with biallelic deletion at 13q14. Two of the three nuclei show no red hybridization signal of a probe containing marker D13S272 demonstrating biallelic loss of this region, while an adjacent probe containing marker D13S273 is retained in disomic fashion. The single cell with two red and two green signals likely represent a non-leukemic cell. (c) Trisomy 12q (three green hybridization signals) and monoallelic deletion 13q14 (single red signal) in two of three nuclei in a B-CLL specimen. A single cell reflecting the normal disomic status of the two regions is shown for comparison.

Molecular genetic techniques

Deletion screening detecting loss of heterozygosity (LOH) by quantitative Southern blot or microsatellite analyses and mutation analyses of genes by single strand conformational polymorphism (SSCP) or DNA sequence analysis have long been limited in CLL due to the lack of candidate genes. Recently, the elucidation of the pathogenic role of p53 and ATM (in a subset of CLL patients) have made molecular genetic screening possible (see below). The identification and sequencing of clonal VDJ rearrangements will be of growing importance to further evaluate the prognostic impact of the VH gene mutational status as biological risk factor in CLL. Therefore, the diagnostic procedure for the detection of mutated VH genes has to become less labor- and cost- intensive. We currently use a set-up consisting of a multiplex PCR from genomic DNA with a mixture of family specific unlabeled 3′-JH primers and fluorochrome-labeled 5′-VH primers. The PCR product is subsequently subjected to a genescan analysis through which the VH gene family involved in the clonal VDJ rearrangement can be identified. The product of the initial multiplex PCR is then directly sequenced with the unlabeled primer corresponding to the VH family involved in the clonal VDJ rearrangement of the respective case.26

Pathogenic and clinical implications of genomic aberrations

Incidence of genomic aberrations in CLL

In conventional chromosome banding studies, trisomy 12 was the first recurrent chromosome aberration described in the late 1970s and early 1980s.10,11,12,13,14 Several investigators confirmed trisomy 12 as frequent aberration in CLL in the following years.27,28,29,30,31,32,33,34,35 Deletions and less frequently translocations involving chromosome band 13q14 were another recurrent aberration identified by several different groups in the late 1980s.32,36,37,38 Further genomic aberrations detected in varying frequencies by conventional cytogenetics included deletions of chromosome bands 11q,29,30,32,37,39 6q14,29,30,34,35 and 17p,34 partial or total trisomy 3q,27,29,32 and translocations involving band 14q32.11,14,27,29,31,34,35,40,41,42 This breakpoint was most frequently the result of a t(11;14)(q13;q32), today considered a hallmark of mantle cell lymphoma (MCL).43 Therefore, many of these cases most likely represented leukemic MCL variants, rather than bona fide CLL cases.

In 1990 and 1991 the largest CLL banding series were reported by the First and Second International Working Party on Chromosomes in CLL (IWCCLL).2,3 Of 662 cases compiled in the Second IWCCLL, 604 were cytogenetically evaluable. Clonal genomic aberrations could be identified in 311 of these CLL cases, with trisomy 12 (19%) being the most frequent abnormality followed by aberrations of chromosome 13 (10%), 14 (8%), 11 (8%), 6 (6%), and 17 (4%).3 However, in 351 cases no clonal abnormality was found.

Due to the methodological problems of conventional chromosome banding studies, it became necessary to reassess the incidence of genomic aberrations in CLL with the aid of novel molecular cytogenetic techniques. Based on conventional cytogenetic analyses and CGH data, a comprehensive DNA probe set was developed allowing the evaluation of the incidence and prognostic significance of the most important CLL-associated genomic aberrations. In our single center study, 325 CLL cases were analyzed by FISH for deletions in the chromosome regions 6q21, 11q22-q23, 13q14, 17p13, for trisomies of bands 3q26, 8q24, 12q13 and for translocations involving the immunoglobulin heavy chain locus on band 14q32. The prevalence of specific genomic aberrations in this large cohort was 82% (268 of 325 cases) and therefore almost twice as high as assumed from chromosome banding studies. The most common aberration was deletion 13q14 (55%), followed by deletion 11q22-q23 (18%), trisomy 12q13 (16%) deletion 17p13 (7%) and deletion 6q21 (7%) (Table 1).4 In multivariate analysis, the 17p and 11q deletions gave significant prognostic information showing that genomic aberrations are important independent predictors of disease progression and survival in CLL (Figure 2a and b).4 However, the prognostic significance of genomic aberrations in CLL has to be confirmed in prospective multicenter controlled treatment trials.

Table 1 Incidence of genomic aberrations in one large retrospective unicentric FISH study compared with preliminary results of two prospective multicenter trials of the German CLL Study Group (GCLLSG)
Figure 2
figure2

Impact of genomic aberrations on the clinical course of CLL. (a) Rate of disease progression as assessed by the treatment-free interval in CLL according to risk groups defined by genomic aberrations.4 The median treatment-free intervals were: 17p deletion, 9 months; 11q deletion, 13 months; 12q trisomy, 33 months; normal karyotype, 49 months; and 13q deletion as single abnormality, 92 months (from Ref. 4 with permission). (b) Survival probability in CLL according to risk groups defined by genomic aberrations.4 The estimated median survival times were: 17p deletion, 32 months; 11q deletion, 79 months; normal karyotype, 111 months; 12q trisomy, 114 months; and 13q deletion as single abnormality, 133 months (from Ref. 4 with permission).

First results from prospective investigations within the CLL1 (fludarabine vs watch and wait in Binet A patients) and CLL3 (high-dose therapy followed by autologous transplantation in Binet B and C patients <60 years) treatment trials of the German CLL Study Group (GCLLSG) are shown in comparison to our unicentric cohort in Table 1.44,45 Regarding the overall incidence of genomic aberrations, these data are consistent with the results from our single center study. The early stage CLL patients entered in the CLL1 trial more frequently show 13q deletions as a single abnormality compatible with a favorable prognosis, whereas in the CLL3 population there is a high incidence of 11q deletions most likely reflecting the preferred inclusion of patients with rapid disease progression. However, among the Binet A patients in the CLL1 trial, 15% show high risk chromosomal aberrations such as deletions 11q or 17p (Table 1).45

Clonal evolution of genomic aberrations has been documented in CLL, however, there are little data so far addressing this topic using molecular cytogenetic techniques. In a conventional cytogenetic analysis carried out by Oscier et al46 karyotypic evolution was seen in 18 out of 112 patients (16%), but there was no correlation between the incidence of clonal evolution and disease progression. In two other chromosome banding studies a significant association between the presence of ongoing karyotype changes and disease progression was seen.47,48 In these investigations 6q and 11q deletions were the most commonly acquired secondary chromosome aberrations associated with a shorter progression-free survival. Using a molecular cytogenetic approach we performed a sequential interphase cytogenetic study applying FISH on 55 CLL patients over a median observation time period of 42 months.49 Clonal evolution was seen in nine out of 55 patients (16%) with 17p deletion (four cases), 6q deletion (three cases), 11q deletion (one case) and evolution from mono- to biallelic 13q deletion (three cases) being the acquired aberrations. We found a significant association between the presence of clonal evolution and progressive disease. Only 20% of the patients with a stable karyotype have died compared to two-thirds of those exhibiting clonal evolution.

In consideration of these studies the sensitive detection of genomic aberrations by interphase FISH provides a basis for a more accurate correlation of genomic aberrations with clinical features in CLL. Interphase FISH and molecular genetic techniques represent excellent tools for a better characterization of the critical genomic regions and have allowed the identification of candidate genes involved in pathogenesis or disease progression of CLL. In the following sections the most frequently involved genomic regions are discussed in detail.

13q14 deletions

Structural aberrations involving the long arm of chromosome 13 were initially reported in smaller chromosome banding studies in the late 1980s.32,35,36,37,38 While in the beginning trisomy 12 was often identified at a higher frequency, in more recent banding series deletions involving 13q turned out to be the most common abnormalities. Most aberrations that involve chromosome band 13q14 are deletions, whereas some appeared as balanced translocations at the resolution power of metaphase chromosome analysis. However, the translocation breakpoints in 13q14 are accompanied by submicroscopic deletions as demonstrated by molecular genetic techniques.50,51

Chromosome band 13q14 harbors the retinoblastoma gene RB1 and this well-known tumor suppressor gene was initially considered to play a pathogenic role in the development of CLL. However, abnormalities disrupting both alleles of RB1 are rarely observed in CLL which argues against the involvement of this gene.50,52,53,54 Because reciprocal translocations involving band 13q14 were more frequently associated with deletions of D13S25 than with RB1, a novel tumor suppressor gene was postulated 1.6 cM distal of RB1 in the genomic region containing the marker D13S25.50,51 The aim of subsequent molecular genetic studies was to define this critical region more precisely.50,51,55,56,57,58,59 Several groups constructed high resolution physical maps spanning several hundred kilobases at the RB1-D13S25 interval.60,61,62,63,64,65 A commonly deleted segment of approximately 300 kb around D13S272 was identified by Kalachikov et al.60 Liu et al61 described a minimally deleted interval of 10 kb centromeric to D13S272. Mutation analysis of two candidate genes in this region, Leu1 and Leu2, today termed BCMS and BCMSUN, respectively, failed to show inactivation of these genes. We constructed a 1.4 Mb sized contig of DNA fragments at the critical D13S273-D13S25 interval and used clones from this to screen 322 B-CLL cases by FISH.64 51% of the examined CLL and 70% of MCL cases showed a 13q14 deletion with a commonly deleted segment involving marker D13S272. However our experiments also did not reveal any evidence for mutational disruption of BCMS or BCMSUN according to the two-hit hypothesis of tumor suppressor gene inactivation. An independently expressed BCMSUN homolog of unclear pathogenic significance was identified in bands 1p22-p31.66 BCMS is organized in a complex fashion spanning more than 560 kb of genomic DNA and is processed into a myriad of transcripts through differential splicing.67 There is currently no evidence for other frequently deleted regions on chromosome 13q, such as the BRCA2 gene in band 13q12,64 therefore, the putative tumor suppressor gene involved in 13q deletions in CLL still remains to be determined.

The clinical significance of 13q14 aberrations was first shown by the multicenter studies of the First and Second IWCCLL, where patients with structural abnormalities of chromosome 13 seemed to have a more favorable prognosis exhibiting survival probabilities similar to those with a normal karyotype.2,3 These findings were supported by the results of our unicentric interphase FISH study.4 The patients with a deletion 13q14 as single aberration (no additional aberration detectable with a comprehensive FISH probe set) had the longest estimated median treatment-free interval and survival time (133 months; see Figure 2a and b). In particular, the estimated median survival time of this group was longer as compared to the groups without detectable aberrations (111 months) and the group with trisomy 12 (114 months).

11q22-q23 deletions

In most chromosome banding studies in CLL, the frequency of 11q22-q23 aberrations has been underestimated. The Second IWCCLL reported abnormalities of the long arm of chromosome 11 in 49 of 604 (8%) cytogenetically evaluable cases.3 Most of these resulted from the translocation t(11;14)(q13;q32) and aberrations involving 11q other than translocations at 11q13 were found in less than 5% of CLL cases. In three more recently published large conventional cytogenetic studies 11q deletion did not occur as frequent aberration.68,69,70 Evidence for the significance of 11q22-q23 aberrations came from smaller chromosome banding studies.29,30,32,37,39,71 In one report, deletion 11q was the second most common chromosomal abnormality identifying a subset of patients who showed rapid disease progression and short survival.72

Regarding the molecular characterization of the critical region affected by deletions of 11q21-q25, only few data were available. One FISH study of diverse hematological neoplasms identified a commonly deleted segment at 11q23.1 containing the neural cell adhesion molecule (NCAM) gene, whereas BCL1 at 11q13 and MLL at 11q23.3 were located outside this critical region.73 For further delineation of the commonly deleted region we performed a molecular cytogenetic study applying FISH in 40 CLL cases,74 using a YAC contig spanning bands 11q14.3-q23.3.75 The minimally deleted region could be narrowed down to 2–3 Mb in bands 11q22.3-q23.1. This finding was later confirmed in an independent series.76 Among the genes contained in this genomic fragment RDX (radixin) and ATM (ataxia telangiectasia mutated) appeared to be the most potential candidate tumor suppressor genes because of their function.77,78 Murine knock-out models gave evidence for a growth suppressor function of ATM as mice deficient for ATM developed T-neoplasms.79 Furthermore, in human T cell prolymphocytic leukemia (T-PLL), ATM deletions and missense mutations leading to disruption of both alleles have been reported.80,81 Absent ATM protein expression in a subset of CLL cases and the description of ATM mutations in a small CLL subgroup made ATM a likely candidate tumor suppressor in this disease as well.82,83,84,85 It was shown that ATM mutant CLL cases exhibited a deficient ATM-dependent response to gamma irradiation, failure to up-regulate TRAIL/R2 and inability to repair induced chromosomal breaks.86 Interestingly, all ATM mutants showed absence of somatic VH hypermutation (see also below) indicating that ATM may play a role at the pre-germinal center stage, where loss of ATM function during B cell maturation may lead to tumorigenesis in pre-germinal cells by a defect in p53 damage response and repair of chromosomal breaks.87 Bullrich et al83 observed ATM mutations not only in neoplastic, but also in normal cells of CLL patients, suggesting the predisposition of heterozygous ATM mutation carriers to develop CLL. However, we were not able to confirm this finding. Furthermore, in our study ATM mutations were only found in five of 22 CLL cases with 11q deletion.85 Therefore, and in contrast to MCL where all cases with loss of 11q show disruption of the remaining ATM allele,88,89,90 the search for additional candidate genes in 11q22-q23 in CLL is ongoing.

First evidence for a prognostic role of 11q aberrations in CLL came from two banding studies showing a correlation between 11q deletions and progressive disease with reduced survival times.71,72 In our large interphase FISH study, we were able to show that CLL patients with 11q abnormalities present with a characteristic clinical picture. Patients with 11q deletion exhibit extensive lymphadenopathy as assessed by the extent of peripheral lymph node involvement and the frequency of mediastinal or abdominal lymphadenopathy and have a more rapid disease progression as shown by a shorter treatment-free interval and reduced overall survival (see Figure 2a and b).91 In multivariate analysis with survival as a dependent variable the presence of 11q deletion gave significant prognostic information.4 Therefore, 11q aberrations identify a new subset of CLL with extensive lymphadenopathy, rapid disease progression and inferior survival. First results from the prospective CLL3 trial of the GCLLSG indicate that deletion 11q23 appears to be associated with an inferior molecular remission rate after high-dose therapy and autografting.44 If this will also translate into inferior clinical outcome remains to be determined by a longer follow-up.

Trisomy 12q13

Trisomy 12 was reported as the first recurrent chromosome aberration in CLL and was the most common chromosome aberration in many chromosome banding studies.13,27,28,29,30,31,32,33,34,35 The frequency ranged from 7% to more than 25% according to the different investigations.92 On banding analysis, only few CLL cases exhibiting a partial trisomy 12 have been identified.16,34,93 Recurrent duplication of chromosome band 12q13-q21.2 was found in all cases, indicating that this region may contain the candidate oncogene(s) playing a pathogenic role in CLL. By restriction length polymorphism (RFLP) analyses, it was shown that trisomy 12 results from duplication of one homolog rather than from loss of one homolog and triplication of the remaining one.94

Trisomy 12 was assessed by interphase FISH by numerous groups,16,68,95,96,97,98,99,100,101,102 determining the frequency of this aberration between 10% and 20% and in two studies from the US even more than 30%.96,98 This variation may be related to patient selection, and may be in some extent due to different geographical distribution of this chromosome aberration as well. However, in all studies directly comparing interphase FISH with conventional cytogenetic chromosome banding techniques, a higher frequency of trisomy 12 was found with the molecular cytogenetic method. In our extended FISH analysis trisomy 12 was only the third most common chromosome aberration seen in 16% of 325 CLL cases.4 Regarding further evaluation of the minimally duplicated segment we were able to identify a CLL case with isolated overrepresentation of a fragment in 12q13-q14.22 Analyzing a complex chromosome 12 rearrangement by FISH, Merup et al103 described a highly amplified region spanning bands 12q13-q15. Another FISH study identified bands 12q13-q22 as minimal duplication segment in B cell non-Hodgkin's lymphoma (NHL).104 So far, no gene from this segment has been proven to be involved in CLL pathogenesis. Recently high resolution mapping of copy number changes using DNA-chip technology became available which may be helpful for narrowing the region of interest.105,106

The clinical significance of trisomy 12 was shown in the First and Second IWCCLL, where patients with this aberration had the shortest survival times among patients with single chromosomal abnormalities.2,3 Some single center banding studies also showed an association with shorter treatment-free intervals and shorter overall survival.30,107 However, this adverse prognostic effect was only shown in univariate analysis and it was not confirmed by other single center chromosome banding analyses.28,29,34,35 Interphase cytogenetics showed an association between trisomy 12 and an increased percentage of atypical lymphocytes or prolymphocytes within the leukemic cell population, as well as an atypical immunophenotype.68,99,100,102 Evaluating the prognostic impact of trisomy 12 by FISH, no significant difference in survival probabilities between patients with or those without trisomy 12 was seen.98 However, when conventional chromosome banding data were included in the analysis of this study the estimated median survival in patients with trisomy 12 was significantly shorter than that in patients with normal karyotype. Furthermore, patients exhibiting an aberration of chromosome 12 were more heavily pretreated and had advanced Binet stages. A sequential FISH analysis of trisomy 12 in CLL showed over a 4-year period similar requirement for treatment and similar overall survival for patients with and without FISH-defined trisomy 12.108 A long-term follow-up of a FISH study that initially reported increased need of therapy and reduced survival in patients with trisomy 12 found no statistical significant difference in survival between patients with and without chromosome 12 aberration after a median observation time of 87 months.109 In our interphase cytogenetic study the estimated median survival time for patients with trisomy 12 was 114 months compared to 111 months in patients with normal karyotype and 108 months for the entire group (see Figure 2a and b).4 On the other hand, preliminary results of the CLL1 trial of the GCLLSG show a significant correlation between trisomy 12 and the high risk group of the study (as determined by diffuse bone marrow infiltration pattern and/or lymphocyte doubling time below 12 months and elevated serum thymidine kinase level and/or elevated serum β2 microglobulin level).45 Whether this association with risk factors indicating rapid disease progression also results into an inferior outcome will be seen in the future.

17p13 deletions and p53 mutations

Structural aberrations of chromosome 17, most commonly resulting from loss of the short arm, were only observed in 4% of CLL cases by the multicenter study of the Second IWCCLL.4 Recently, another large chromosome banding study confirmed the incidence of 4% for chromosome 17 abnormalities in CLL as detected by conventional cytogenetics.69 Evidence for more frequent disruption of chromosome 17 leading to the loss of the short arm came from smaller banding series.34 However, the pathogenic role of 17p13 deletions, the location of the tumor suppressor gene p53, became more evident through molecular genetic studies investigating this candidate gene. Gaidano et al110 found p53 mutations in six of 40 (15%) CLL cases by SSCP analysis and sequencing of the PCR amplified fragments. Subsequent studies also reported p53 mutations at frequencies ranging from 10% to 15%.111,112,113 Based on these molecular genetic findings we applied FISH using a p53 containing genomic probe to screen a large series of CLL patients for 17p13 deletions. In our initial series of 100 cases we found p53 deletions at an incidence of 17%,114 whereas our extended series showed only an incidence of 7%.4 This difference is most likely due to patient selection as the initial study also included cases of B-PLL which showed a high frequency of 17p deletions. In line with the two-hit hypothesis we were able to demonstrate a disruption of the remaining p53 allele by mutation in most cases leading to inactivation of this recessively acting tumor suppressor gene.115 Until recently, p53 was the only gene shown to be involved in the pathogenesis of CLL, and it is interesting to note that ATM mutations which are found in a subset of CLL cases with 11q deletion can also result into dysfunction of the p53 pathway.87

A strong prognostic impact of p53 abnormalities was demonstrated in the study by El-Rouby et al,112 who showed that p53 abnormalities predicted for treatment failure with alkylating agents. A recent banding analysis of 480 untreated CLL patients within a randomized trial of alkylator therapy found abnormalities involving 17p13 to be the only chromosomal aberration of prognostic significance.69 Our initial interphase cytogenetic study on 100 CLL cases identified 17 patients exhibiting a 17p deletion whose clinical course was characterized by significantly shorter survival times compared to patients without this aberration.114 In addition, patients with 17p deletion showed no response to therapy with purine analogs. Whereas 56% of patients without p53 disruption responded to treatment with fludarabine, none of the patients with a p53 deletion did. In multivariate analysis p53 deletion was revealed as the strongest prognostic factor (see Figure 2a and b).4 A recent study on 122 CLL patients also showed that p53 abnormalities are more common in refractory advanced disease and that p53 aberrations are associated with treatment resistance and shorter survival.116 There is anecdotal evidence that a response may be achieved in CLL with p53 inactivation with the monoclonal anti-CD52 antibody Campath-1H.117

6q21 deletions

Aberrations of the long arm of chromosome 6 are among the most common chromosomal abnormalities in lymphoid neoplasms.118 The Second IWCCLL found structural aberrations of chromosome 6 in 6% of the evaluable tumors and described chromosome bands 6q15 and 6q23 as most commonly involved.3 Based on molecular genetic analysis of several subtypes of malignant lymphomas, at least two independent regions of commonly deleted segments, one at 6q21-q23 and one at 6q25-q27, have been identified.119 In small lymphocytic lymphoma (SLL), the lymphomatous counterpart of CLL, especially deletions of 6q21-q23 were identified.120 Recently, in CLL the proximal location of the minimally deleted region was confirmed by Merup et al,121 who identified a critical deletion region spanning markers D6S283 to D6S270 on chromosome band 6q21 in 6% of CLL cases. Our investigations with FISH using DNA probes mapping on 6q21 and 6q27 confirmed these findings showing that all deletions can be detected with the 6q21 probe, whereas only one-third of the patients also showed 6q27 deletions.122 Zhang et al123 determined a 4–5 Mb minimal deletion region in band 6q21 in a variety of lymphomas and lymphoid malignancies. Several genes in this region have been identified, but none of these has so far been shown to play a role in the pathogenesis of CLL.

Patients with deletion of the long arm of chromosome 6 were shown to have shorter treatment-free intervals in one single center study.35 In patients with follicular NHL deletion 6q has also been identified as the negative prognostic factor.124 On the other hand, the two large multicenter studies of the IWCCLL did not observe any adverse prognostic impact of 6q deletions.2,3 In our interphase cytogenetic study 6q deletion was associated with a higher tumor mass as represented by higher white blood cell counts and more extensive lymphadenopathy, but the median treatment-free intervals and overall survival times were not significantly different between the groups with or without 6q deletions.122

14q32 (IgH) translocations

The Second IWCCLL reported aberrations of chromosome 14 clustering in band 14q32, the locus of the immunoglobulin heavy chain (IgH) gene, in 8% of evaluable cases.3

t(11;14)(q13;q32):

The translocation t(11;14)(q13;q32) is leading to the fusion of the BCL1 locus at 11q13 to IgH at 14q32 resulting in overexpression of cyclin D1 (CCND1).125,126,127,128,129,130 However, this translocation today is considered a hallmark of MCL and may occur at low frequencies in other lymphoproliferative disorders distinct from CLL.43,125,126,127 Although early molecular studies suggested a pathogenic role of CCND1 in CLL,128,129,130 a re-evaluation classified the examined cases as MCL.43 Recent analyses showed no evidence for a frequent involvement of CCND1 in CLL.4,113,131,132,133,134

t(14;18)(q32;q21):

Translocations involving BCL2 at 18q21 were also suggested to play a pathogenic role in CLL.135,136 The t(14;18)(q32;q21) is characteristic of follicular NHL, but overexpression of BCL2 is also observed in other lymphoid malignancies, such as CLL. In follicular lymphoma breakpoints of t(14;18) occur in the major breakpoint region (mbr) or in the minor cluster region (mcr) at the 3′ end of BCL2, whereas in CLL the breakpoints have been found to be at the 5′ end juxtaposing BCL2 to the immunoglobulin light chains. However, large series showed BCL2 rearrangements to be rare events in CLL.4,113,133,137,138,139

t(14;19)(q32;q13):

From the 19q13 breakpoint of this translocation the BCL3 gene was cloned.140,141,142,143,144 Rearrangement involving the BCL3 gene locus at 19q13 were found in six out of 4487 cytogenetically analyzed lymphoproliferative disorders with five of the six cases classified as CLL.4,140

In the light of large studies of well classified tumors the previously described ‘14q+ marker’ does not appear to be a frequent aberration of CLL. Rearrangements of the IgH locus with oncogenes appear to be rare events in CLL.145

Infrequent genomic aberrations

Other prominent cancer genes such as p16 (CDKN2A) located in chromosome band 9p21, frequently involved in other types of neoplasms,146,147,148 do not appear to have a role in the pathogenesis of CLL.149 Chromosome banding analyses and CGH studies also reported other recurrently involved genomic regions in CLL. These abnormalities are commonly gains of chromosomal regions, such as trisomy 3.27,29,32 CGH data suggested that the minimally duplicated segment comprised the distal region of the long arm, which could be a locus for an oncogene of possible pathogenic significance in CLL.22 CGH data also revealed another region of interest, gains of the long arm of chromosome 8.22 Automated genomic profiling using microarray based hybridization (matrix-CGH) may be a new powerful technique for detection of genomic aberrations in lymphoid malignancies.150 Preliminary analyses in CLL identified additional genomic gains in bands 2q22, 7q31, and 11q25.151

Pathogenic and clinical implications of VH gene mutations

Characterization of VH gene mutations in CLL

Based on the expression of CD5, CLL cells appeared to correspond phenotypically to mantle-zone-derived, naive B cells. Early studies confirmed the expectation that no somatic mutation would be found in the VH genes of CLL cells.152,153,154 However, an extensive review of Schroeder and Dighiero6 showed that approximately half of 75 reported CLL immunoglobulin VH gene sequences varied by more than 2% in sequence similarity as compared to the nearest germline gene, consistent with somatic hypermutation. In a subsequent study, Fais et al7 found that approximately 50% of IgM+ CLL cases and approximately 75% of IgG+ and IgA+ cases showed significant VH gene mutations. The degree of VH gene mutation in these CLL cells was considerable, since more than a third of the IgM+ CLL cells and more than two-thirds of the isotype-switched CLL tumors showed 5% differences from their most similar germline genes. Based on these data, CLL can be segregated in two subgroups on the basis of the presence or absence of significant numbers of VH gene of mutations.7,8 The discrepancy between initial studies of VH mutations in CLL and the recent results may be due to the observation that VH gene mutations are distributed nonstochastically and appear to relate to the VH family gene expressed in the CLL cell.7 A VH family-related hierarchy of mutation (VH3 > VH4 > VH1), which was most obvious when comparing the VH 3–07 (90% of cases mutated), VH 4–34 (73%), and VH 1–69 (17%) genes, was reported by Fais et al.7 Therefore, small patient cohorts with nonrandom VH gene family expression may demonstrate different levels of somatic mutation. The reason for the VH family-related hierarchy of mutation in CLL is still unclear, but might reflect differences in the types of antigens that have driven the individual B cells before their leukemic transformation and/or differences in the maturation stages at which they were transformed into leukemic cells.7,8,9,155 In addition to the overrepresentation of VH3 and VH4 family gene members in VH mutated cases and overrepresentation of VH1 family genes in the VH unmutated group, there is also a difference in JH gene usage and in the complementary determining region (CDR3) structure reflected by CDR3 length and D segment sequence conservation rates between the two subgroups of CLL.7,156,157 However, to date the biological significance of these findings is still unknown.

B cell tumors have recently been classified in three categories: (1) those with unmutated VH genes, in which it was postulated that the cell of origin had not entered the germinal center; (2) tumors with ongoing VH gene mutations, such as follicle center lymphoma, in which it was postulated that the malignant cells remain under the influence of the germinal center reaction; and (3) B cell tumors with mutated stable VH genes, such as multiple myeloma, which were postulated to have irreversibly traversed the germinal center.158 Therefore, the finding that CLL can either exhibit VH gene sequences in germline configuration, or with somatically mutated VH genes, as independently reported in several studies,7,8,9,159 illustrates a novel scenario with a single tumor entity being composed of different subtypes, with regard to the germinal center reaction of VH gene hypermutation. In addition and in contrast to FCL, the mutational pattern in CLL appears stable and does not show any intraclonal heterogeneity.7,8,9,158,159,160

Correlation of VH gene mutational status with BCL6 mutations

Somatic hypermutation can also be observed in non-immunoglobulin genes as evidenced in the 5′-intronic region of the BCL6 gene.161 BCL6 is a proto-oncogene mapping to chromosome 3q27,162 which encodes a transcription protein with a POZ/Zinc finger motif.163 In NHL, a high frequency of chromosomal translocations cluster at band 3q27, specifically at the BCL6 promoter, which might lead to deregulated BCL6 function with a potential role in lymphomagenesis.164,165,166 However, mutation events at this locus also occur in the absence of translocations.161 In both, the VH and BCL6 loci, somatic mutation is only found in memory and not in naive cells in normal B cells.167,168,169 At the single cell level, BCL6 mutations tended to occur in normal B cells containing VH gene mutations.167 In addition, one study reported that the occurrence of BCL6 mutations in eight out of 34 CLL cases was restricted to VH mutated cases, supporting the hypothesis that BCL6 mutations result from the same process that targets immunoglobulin genes.170 However, in the initial study, 80% to 100% of the cells displayed VH gene mutations, whereas only approximately 30% of these cells had corresponding mutations in BCL6, indicating different targeting of the two loci.167 In CLL two studies were able to identify somatic mutation not only in the cases with mutated VH genes, but also in cases with no VH mutations.171,172 One further unexpected feature was intraclonal variation in BCL6 evident in six out of eight mutated cases.172 As in CLL intraclonal heterogeneity in VH is very rare, it was not found in the VH genes of the cases with heterogeneity in BCL6, suggesting that the mutation mechanism may operate differently on the two loci. Recently, ongoing mutations in CLL were also described in the CD79 genes, regardless of the mutational status of the corresponding VH gene.173 The pathogenic role of mutations in the non-coding region of BCL6 is so far unknown and in contrast to the VH status BCL6 mutations do not apparently correlate with prognosis.172

Correlation of VH gene mutational status with CD38 expression

It still remains unclear whether CLL cells with unmutated VH genes are naive B cells or represent B cells that have been antigen-stimulated, but have not accumulated mutations. To more accurately define the stage of maturation at which the CLL cells are arrested, Damle et al8 studied the surface membrane expression of CD38 and IgD on CD5+/CD19+ B cells in a series of well-characterized cases. Analyses of these surface markers have been especially useful in distinguishing B cells at various stages of differentiation from naive through memory cells.174 Damle et al8 found a heterogeneous CD38 expression in CLL with some cases showing an expression of CD38 on almost 100% of the malignant clone, whereas in other cases only few, if any leukemic cells expressed CD38. Based on the percentage of clonal cells expressing CD38, CLL could be subdivided in two categories: one with <30% CD38+ CLL cells and another with 30% CD38+ CLL cells.

The analysis of Damle et al8 showed that the set with a higher percentages of CD38+ cells (30% CD38+ CLL cells) was comprised of unmutated CLL cases, whereas the set with lower percentages (<30% CD38+ CLL cells) contained almost exclusively the mutated cases indicating a strong inverse relationship between VH gene mutation and CD38 expression. CD38 expression was also reported to be stable over time and it was not influenced by chemotherapy in this study. However, other studies failed to find this strong correlation between unmutated VH genes and high CD38 expression, which is therefore currently a matter of discussion.175,176,177 In agreement with the above-mentioned studies, we also observed an inverse correlation between VH mutational status and CD38 expression, but in approximately one-third of cases CD38 expression failed to predict the VH mutational status.26 The variation observed among different studies might be due to methodological aspects, but with the current knowledge CD38 expression appears not to serve as a valid surrogate marker for the VH gene mutational status in CLL.

Correlation of VH gene mutational status with genomic aberrations

With the VH gene mutation status and genomic aberrations, there are two genetic parameters among the most powerful risk factors in CLL, but the relation of these two has only recently been evaluated (Table 2).26,178 The overall incidence of aberrations was not different between the VH mutated and unmutated subgroups in our study (80% vs 84%). However, there was a significant difference in the incidence of high risk aberrations deletion 17p (3% vs 10%) and deletion 11q (4% vs 27%), as well as a difference in the incidence of favorable aberrations such as deletion 13q (65% vs 48%) and del(13q14) as single aberration (50% vs 26%).26,178 In a smaller interphase cytogenetic study, our finding that genomic aberrations are seen in the VH mutated, as well as in the unmutated subgroup were confirmed, but no association between specific aberrations and the VH gene mutational status was observed possibly due to the small patient number.179 The difference compared with the results of Oscier et al159 and Hamblin et al9 who observed more cases exhibiting trisomy 12 in the VH unmutated subgroup is most likely attributable to different technology used. Chromosome banding was performed in the Oscier/Hamblin study, while we applied FISH, a much more sensitive technique to detect genomic aberrations in CLL. This is underlined by the observation that by banding generally a lower incidence of aberrations is detectable in particular in VH mutated CLL possibly due to the generally lower in vitro proliferative potential of the VH mutated CLL cells.9,159

Table 2 Correlation of VH mutation status with genomic aberrations in 300 CLL cases

Clinical impact of VH gene mutation status, CD38 expression and genomic aberrations

The pioneer studies by Damle et al8 and Hamblin et al9 reported a significant correlation of the VH gene mutational status with disease course and survival in CLL. Collectively, the two studies reported results on 131 CLL patients showing that cases with a VH gene homology 98% (ie unmutated VH) experienced a more aggressive clinical course with shorter survival times compared to cases with homology <98% (ie mutated VH). The estimated median survival time in the study by Damle et al8 was in the unmutated group 108 months and not reached in the mutated group, whereas Hamblin et al9 reported 117 months in unmutated and 293 months in unmutated patients, respectively. The latter study also showed this correlation in Binet A patients with an estimated median survival time of 95 months for patients without mutation and 293 months for those with mutations. Subsequent studies confirmed the prognostic impact of the VH gene mutational status on survival.176,180

Analogous to the pivotal studies, our patients with mutated VH genes had significantly higher survival probabilities compared to the patients with unmutated VH genes.26,178 Interestingly, in our series the estimated VH homology rate yielding the best separation of two subgroups with different survival probabilities was not the classical cut-off value of 98% but 97% VH homology to the nearest germline gene (Figure 3a and b). This finding was derived from careful statistical analysis of survival time in relation to VH homology, instead of applying the classical 98% cut-off value for the definition of mutated VH derived from germline polymorphism considerations.26 Further studies are needed to determine the biological significance of this finding. Furthermore, first data examining the influence of the VH mutation status on outcome after specific treatment modalities are becoming available. The group of Dreger et al has recently demonstrated that the adverse prognostic influence of unmutated VH may be retained even after aggressive treatment, such as high-dose therapy and autologous stem cell transplantation.181

Figure 3
figure3

Probability of survival from the date of diagnosis among patients with mutated and unmutated VH genes according to the 97% and the 98% cut-off values. (a) The estimated median survival time for the VH homology 97% group was 79 months. The last observed death in the VH homology <97% group was after 152 months of follow-up time (survival probability 56%). (b) When only patients diagnosed at Binet stage A were evaluated, the estimated median survival times for the VH homology 97% and VH homology <97% groups were 79 months vs not reached (last observed death after 152 months of follow-up time; survival probability 53%).

Damle et al8 initially demonstrated the CD38 expression level as significant prognostic marker in CLL. The estimated median survival time for patients with 30% CD38+ CLL cells was 120 months in this study, as compared to the group of patients with lower CD38 expression level, in which the estimated median survival time was not yet reached. Subsequent studies confirmed the prognostic value of the CD38 expression level.177,182,183 However, our investigations in 157 CLL cases and a study by Thunberg et al, failed to show a significant difference in survival probability when using the 30% cut-off for CD38 positivity.26,176 By maximally selected log rank statistics, the best separation of two subgroups with different survival probability was achieved for a cut-off value of 7% CD38-positive CLL cells in our study. The estimated median survival times for the group with <7% CD38+ clonal cells was 79 months, and 114 months for the group with <7% CD38+ clonal cells.26,178

For the evaluation of the relative prognostic impact of the VH mutation status, CD38 expression level, genomic aberrations, clinical, and laboratory parameters we performed a multivariate analysis.26,178 Unmutated VH, 17p deletion, age, white blood cell count, and serum lactate dehydrogenase were identified as significant prognostic factors at the 97% VH homology cut-off, while at the 98% cut-off 11q deletion entered the model as additional independent prognostic factor. The hazard ratios together with their 95% confidence intervals are shown in Table 3. Of note is the fact that clinical staging according to Binet provided a significant separation of subgroups with respect to their survival time distributions, but was not an independent prognostic factor in the knowledge of VH mutation and 17p deletion status. This observation is illustrated in Figure 4 showing the survival probabilities in the dominant genetic subgroups for all (a), and for Binet-A patients (b). In conclusion, it appears that with the VH mutation status and genomic aberrations, parameters became available which may allow a risk assessment of CLL patients at the time of diagnosis independently of the stage of their disease.

Table 3 Cox regression analysis of survival time from diagnosis in 300 cases of CLL26
Figure 4
figure4

Probability of survival among patients in the following genetic categories: 17p− (17p deletion irrespective of VH mutation status), 11q− (11q deletion irrespective of VH mutation status), unmutated VH (VH homology 98% and no 17p or 11q deletion), and mutated VH (VH homology <98% and no 17p or 11q deletion). (a) Among the entire cohort (n = 300), the estimated median survival times for the respective genetic subgroups were: 17p deletion, 30 months; 11q deletion, 70 months; VH unmutated, 89 months; and VH mutated; not reached (54% survival at 152 months). (b) Among Binet A patients (n = 189) the estimated median survival times for the respective genetic subgroups were: 17p deletion, 36 months; 11q deletion, 68 months; VH unmutated, 86 months; and VH mutated, not reached (52% survival at 152 months).

Perspectives

Over the past decade, rapid progress has been made in the genetic analysis of CLL. With the aid of interphase cytogenetics recurrent genomic aberrations are observed in more than 80% of cases and with ATM and p53 genes involved in disease pathogenesis and progression were identified. However, for the most frequent genomic abnormalities candidate genes still have to be isolated. The discovery of the two CLL subtypes as defined by the mutation status of the VH genes has further highlighted the molecular heterogeneity of the disease. Novel tools such as DNA microarrays already had strong impact on the further clarification of the molecular background of CLL.151,184,185,186 In particular, these studies have shown that both the VH mutated and the unmutated type of CLL show gene expression patterns similar to memory B-cells and have therefore shed doubt on the theory that CLL with unmutated VH genes is derived from naïve pre-germinal center lymphocytes. Furthermore, genetic parameters have shown their prognostic value in the identification of CLL patients who are at risk for rapid disease progression, resistance to therapy and short survival. Particularly, unmutated VH genes, 11q and 17p deletions were among the strongest independent risk factors and appear to identify patients at risk for poor outcome irrespectively of their clinical stage. Therefore, at the time of diagnosis a “state of the art risk assessment” should today ideally include a comprehensive genomic screening, an evaluation of the VH mutation status and CD38 expression level, in addition to other well known clinical and biological parameters. Further evaluation of these prognostic markers within prospective treatment trials is needed to give us the opportunity for a more refined disease management in the future, especially with regard to the availability of highly effective treatment approaches such as purine analogs, antibodies and autologous or allogeneic stem cell transplantation.

Editor's note

We are very indebted to Dr Peter Daniel who recruited and evaluated all the Reviews published in this Spotlight. Authors who are interested in contributing a Review for this Spotlight are invited to contact the Editor-in-Chief, Dr Muller Bérat.

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Acknowledgements

This work was supported by the Wilhelm Sander-Stiftung (No. 2001.004.1), University of Ulm (No. P.679), Deutsche Krebshilfe (No. 10–1289-StI), and BMBF (Nos. 01KW9934, 01KW9938).

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Correspondence to H Döhner.

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Stilgenbauer, S., Bullinger, L., Lichter, P. et al. Genetics of chronic lymphocytic leukemia: genomic aberrations and VH gene mutation status in pathogenesis and clinical course. Leukemia 16, 993–1007 (2002) doi:10.1038/sj.leu.2402537

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Keywords

  • CLL
  • genomic aberrations
  • p53
  • ATM
  • VHmutation status

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