SPOTLIGHT 18 of the ﬁrst Draft of the Human Genome, Identiﬁcation of the the Nobel Prize for Medicine in 1987

The conﬁguration of the immunoglobulin genes in B cell chronic lymphocytic leukemia MJS Dyer1 and DG Oscier2

1Department of Haematology, University of Leicester, Leicester, UK; and 2Department of Haematology, Royal Bournemouth Hospital, Bournemouth, UK

B cell chronic lymphocytic leukemia (CLL) lacks a consistent associated with loss of expression of some genes, but possibly genetic abnormality. However, immunoglobulin VH gene seg- over-expression of others.8 ment mutation analysis has provided insights into the pathogenesis of these diseases and allowed the development of Given these difficulties, an alternative approach has been powerful prognostic markers. Immunoglobulin gene to search for abnormalities within genes of importance in the chromosomal translocations are rare in CLL and involve a dis- pathogenesis of other subtypes of B cell malignancy. The B tinct subset of genes including BCL3, BCL11A and CCND2. cell non-Hodgkin’s lymphomas (B-NHL) are in many BCL2 translocations in CLL appear to arise via a different instances, characterized by specific and recurrent chromo- mechanism from comparable translocations seen in B cell non- somal translocations involving the immunoglobulin (IG) Hodgkin lymphoma. 9–11 Leukemia (2002) 16, 973–984. DOI: 10.1038/sj/leu/2402528 loci. Molecular cloning of such translocations has allowed, Keywords: chronic lymphocytic leukemia; immunoglobulin genes; and continues to allow, cloning of genes intimately involved VH mutations; BCL6 mutations; chromosomal translocations in the development of both normal and malignant B cells.12–15 These genes may be involved in the pathogenesis of B cell malignancies, not only through chromosomal translocation, Introduction but also through their direct involvement in genomic amplifications and through changes in methylation. Moreover, Despite much work, the molecular pathogenesis of B cell molecular analysis of the breakpoint sequences allows identi- chronic lymphocytic leukemia (CLL) remains obscure. The fication of the mechanisms and timing of chromosomal trans- 10,16 classical cytogenetic-lead approach to the molecular dissec- locations in B cell development. tion of malignancy has thus far failed to allow the identifi- Here, we review data concerning the configuration of the cation of the major ‘players’. The reasons for this reflect not IG genes in CLL and how studies of these genes have shed only the difficulties in obtaining metaphases from patients light on the pathogenesis and progression of CLL. with CLL, but also the lack of a consistent and a genetically amenable lesion. Cytogenetic analysis of CLL supplemented with comparative genomic hybridization and interphase FISH Somatic recombination and hypermutation of the IG genes studies, have shown recurrent deletions involving regions in normal B cells such as 6q21, 11q22-q23, 13q14.3 and 17p13.1.1 According to the Knudson hypothesis, such deletions may mark the pres- The origins of antibody diversity reflect the actions of three ence of tumor suppressor genes (TSGs) with deletion of one fascinating genetic mechanisms summarized with reference to allele and mutation of the other. Whilst this hypothesis has the immunoglobulin heavy chain (IGH) locus at the very telo- worked well in the isolation of inherited cancer genes, the mere of the long arm of chromosome 14 at chromosome number of somatically mutated TSGs remains small. There are 14q32.33 in Figure 1 and reviewed in Refs 10 and 17 and a variety of possible reasons for this.2 Firstly, somatically Refs therein. acquired deletions are frequently large, spanning several Firstly, somatic recombination of the dispersed IG gene seg- megabases of DNA and may be secondary events, present ments is required to produce a functional IG gene. This was only in a fraction of malignant cells. Even with the completion first demonstrated by Susumu Tonegawa, for which he won SPOTLIGHT 18 of the first draft of the human genome, identification of the the Nobel prize for Medicine in 1987. During the early key gene within the involved region (such as the commonly stages of B cell differentiation within the bone marrow, diver- deleted region of 13q14 in CLL) may therefore be extremely sity (D) and then variable (V) region gene segments undergo difficult.3–5 Alternatively, for some genes, loss of one copy DNA sequence specific recombination with the joining (J) without mutation of the remaining allele (haploinsufficiency) region segments in order to produce a contiguous VDJ gene. may be sufficient, whilst for other TSGs, epigenetic factors, This is mediated by a complex of enzymes, including RAG1 such as promoter methylation may also contribute to loss of and RAG2, which bind to and cleave specific DNA expression.6,7 Furthermore, in CLL, some 13q14 deletions may sequences. Part of the genetic diversity arises through the dif- in fact be associated with chromosomal translocations, sug- ferent junctions arising from the assortment of the various V, D gesting that some of these events are complex and may be and J segments. Cell surface expression of functional antibody molecules is necessary for the continued survival of early pre- B cells. Failure to undergo recombination successfully due to out-of-frame recombination, results in cell death by apoptosis. Correspondence: MJS Dyer, Department of Haematology, University The dependence of B cell survival on maintained surface Ig of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester 19 Royal Infirmary, PO Box 65, Leicester, LE2 7LX, UK; Fax: 44-116- expression persists until differentiation to memory B cells. 252-3274 After successful recombination, expression of the Received 27 November 2001; accepted 19 February 2002 rearranged VDJH sequences is initially driven by the proximity Configuration of immunoglobulin genes in B cell CLL MJS Dyer and DG Oscier 974 SPOTLIGHT

Figure 1 Schema showing the various conﬁgurations of the immunoglobulin heavy chain (IGH) locus on chromosome 14 during B cell

differentiation. The IGH locus is located at the very telomere of the long arm of chromosome 14. It comprises 52 dispersed variable (VH), 27 diversity (DH) and six joining region (JH) segments. VDJH recombination occurs within the bone marrow and results in the formation of a contiguous gene segment. SHM and CSR occur in the germinal center within the lymph node via a related mechanism. Both involve double- stranded DNA breaks. Switch regions are shown in gold, constant regions in dark blue and enhancer regions in purple. Enhancer regions are ␮ ′ ␣ ␣ ␦ ␥ located in the JH-C intron and 3 of both C 1 and C 2; recently an additional enhancer region has been identiﬁed between C and C 3. Transcriptional orientation is shown by horizontal arrows.

of the VH promoter to a transcriptional enhancer located some regions are hot spots for SHM are incompletely under- ␮ 28,29 within the intron between the JH segments and C , immedi- stood. ately upstream of the repetitive S␮ region. The anatomy of this In normal B cells, replacement mutations are clustered enhancer, in terms of definition of protein binding sites, has within the CDRs that encode the antibody-combining site, been extensively studied. Apart from its profound effects in rather than the intervening framework regions (FR), which are directing B cell-specific transcription, this small region of responsible for the structural integrity of the variable region. DNA has a number of other interesting properties. It also The ratio of replacement mutations (R) resulting in a change appears to act as an attachment site to the nuclear matrix and in amino-acid content to silent mutations (S), which do not is, as well, an origin of DNA replication.20–22 The intronic affect the protein structure, is therefore higher in CDR than in enhancer of both IG heavy and light chains is necessary for FW regions. The consequence of SHM is the selection of B somatic hypermutation to occur.23 Properties of the enhancer cells that produce high affinity antibodies. Mutated cells that may also account for the differential subnuclear localization produce low affinity antibody or fail to produce antibody, like of the two IGH alleles observed in normal B cells and may the equivalent B cell precursors in the bone marrow, undergo account in part for the allelic exclusion of immunoglobulin apoptosis within the germinal center. The finding of intra- proteins.24 Interestingly, another IG enhancer or possibly an clonal heterogeneity in normal germinal centre B cells indi- IGH locus control region (LCR) has been recently identified cates that these cells may undergo more than one cycle of in a highly unstable region of genomic DNA between the C␦ SHM. and the first downstream C␥ gene, C␥3. This appears to func- Finally, shortly after SHM occurs within the germinal tion in the control of early B cell development although its center,30 the third recombination process, involving transpo- 25 role in mature B cell malignancies has not been investigated. sition of the completed VDJ gene to downstream constant (CH) This region may also contain a number of nuclear matrix region segments is initiated resulting in antibody molecules attachment regions. Breaks in this region are seen in at least with different effector functions. This is known as class switch 10% of cases of B cell precursor ALL, but comparable abnor- recombination (CSR). CSR occurs between highly repetitive malities in CLL have not been detected.26 switch regions, which lie upstream of each of the IG constant Secondly, somatic hypermutation (SHM) is necessary to region genes with the exception of C␦. However, despite the produce antibodies with high affinity for antigen. This occurs highly repetitive nature of individual switch regions, CSR does primarily, but not exclusively, at the complementarity not represent homologous recombination. The discovery that determining regions (CDRs), the antigen-binding sites of the an enzyme with homology to RNA editing enzymes human

VH genes. This process occurs within the germinal center fol- activation-induced cytidine deaminase (AID) is involved in lowing encounter with antigen. Cells expressing high-afﬁnity both SHM and CSR indicates some common pathways in antibody molecules are positively selected and differentiate both processes.31 into either memory B cells or plasma cells. Once selected, All three recombination processes, including SHM, involve memory B cells no longer require surface immunoglobulin or the introduction of double-stranded DNA breaks.32,33 It has antigen for continued long-term survival.27 The signals that thus been suggested that errors in all three processes may con- trigger SHM, the mechanism responsible and the reasons why tribute to the pathogenesis of B cell malignancies in various

Leukemia Conﬁguration of immunoglobulin genes in B cell CLL MJS Dyer and DG Oscier

Ͻ 975 ways, but principally through the generation of chromo- between different VH gene segments. Although 98% hom- somal translocations.10,32 ology to the nearest germline sequence is regarded as evi- A final point of interest concerning the immunoglobulin dence of SHM, this figure can only be an approximation until genes is that mature B cells express only one allele, a process all the polymorphisms in every VH gene segment are known. known as allelic exclusion. In some instances, only one allele Initially VH gene sequencing was performed on gel-purified may be functional due to incorrect VDJ rearrangement PCR products, which were then cloned into bacterial vectors. resulting in an out-of-frame join. In others though, it is clear Sequencing a large number of clones from a single patient that both alleles are potentially functional. Nevertheless in all enables the incidence of intra-clonal heterogeneity to be normal cells, and in the majority of neoplastic B cells it seems determined. In CLL, this occurs only rarely and it is now more that only one allele is expressed. In a subgroup of CLL, it has usual to sequence PCR products directly. However, it is poss- been shown that this process is subverted with the simul- ible that very small intraclonal variations may be missed using taneous expression of two IGH alleles.34,35 This appears to direct sequencing. Most sequence data have been derived 40 occur quite frequently, in 8/122 (6%) of cases in one series. from analysis of VH genes only. Damle et al have also The possible biological and clinical significance of this obser- sequenced VL genes and found that in 5% of CLL cases vation is not known. Interestingly, allelic exclusion is also mutations are restricted to the VL genes. subverted in all cases with IG translocations since both the Early studies on small numbers of cases showed that VH functional IG allele and the translocated oncogene at the other genes were unmutated in CLL.41 This was consistent with the IG allele are expressed simultaneously. Allelic exclusion may hypothesis that CLL, together with mantle cell lymphoma, was result from the differential methylation of the IGH loci, and derived from a subset of CD5 positive naive B cells. In 1994, 42 this may account for the differential nuclear sublocalization Schroeder and Dighiero reviewed the literature on VH gene of the two alleles.24,36 status in CLL and noted that 36 of 75 cases had Ͻ98% hom- In health, 60% of circulating B cells in the peripheral blood ology to the nearest germline sequence. The presence of are naı¨ve pre-germinal center cells which express surface IgM mutations in 40–70% of patients with CLL has subsequently and IgD but not CD27, and have unmutated VH genes; 25% of been confirmed in a number of series that have investigated these are CD5+. The remaining cells are post germinal center over 700 patients in total (Table 1). The variation in the inci- memory cells, which have undergone SHM and which dence of cases with SHM largely reflects the clinical stage of express CD27. Approximately 40% are class switched, while patients analyzed. In the Bournemouth series, 49% of Binet the remainder express surface IgM and/or IgD, but not CD5.37 stage A patients and 69% of stage B or Cpatients had un- 43 mutated VH genes. This difference is the probable explanation for the initial finding of unmutated VH genes in CLL Analysis of the IG genes in B-CLL since the cases selected for study were likely to have had advanced disease, emphasizing the dangers of extrapolating VH mutations in CLL data on small numbers of cases to a whole disease.

VH gene analysis is increasingly being employed in the investi- 38 gation of chronic B cell malignancies. These may be classi- CD38: a surrogate marker for VH mutational status ﬁed as pre-germinal center, germinal center or post germinal center tumors depending on whether SM has occurred and if CD38 is a trans-membrane glycoprotein which is widely so whether there is intraclonal heterogeneity. However un- expressed on hemopoietic cells, but is expressed at high levels expected heterogeneity in VH gene status is being encountered on germinal center B cells and plasma cells. CD38 is a novel in several B cell lymphomas that appear histologically enzyme capable of catalysing multiple reactions, including uniform.39 NAD glycohydrolase, ADP-ribosyl cyclase, cyclic ADP ribose

Technically, VH gene analysis may be performed on gen- hydrolase and base-exchange activities. The functions of the omic DNA or cDNA. The advantage of using cDNA is that molecule are complex and multiple, but germinal center B only the functional gene product is sequenced. If genomic cells are rescued from apoptosis by CD38-mediated signaling. DNA is used, the non-functional rearrangement is often The pathway used is the same as that used for B cell receptor detectable and also undergoes SHM in cells which have tra- for antigen/Ig signaling (reviewed in Ref. 44). versed the germinal center. PCR primers are usually selected Damle et al40 observed that Ͼ30% expression of CD38 on SPOTLIGHT + + such that the whole of the VH,DH and JH gene segments are CD5 /CD19 CLL cells was strongly associated with unmuat- ampliﬁed. Mutations are counted within the VH gene from the beginning of framework 1 to the end of framework 3. The Table 1 Summary of studies on VH gene mutation status in CLL presence of N additions between the VH and DH segments and the DH and JH segments precludes mutations within the CDR3 Study (Ref.) Year No. of % % region from being counted. If a primer within the framework cases Mutated Unmutated 1 region is used rather than in the VH gene leader sequence, the number of nucleotides that can be assessed for the pres- Schroeder/Dighiero42 1994 75 48 52 ence of mutations is reduced and as mutations are rarely Fais et al60 1998 64 sIgM+ 50 50 19 sIgM− 75 25 found within framework 1 the percentage of VH mutations 43 may appear slightly higher. Assignment of a nucleotide Hamblin et al 1999 84 54 45 Maloum et al49 2000 39 69 31 change as a mutation rather than a polymorphism depends on Matrai et al48 2001 40 40 60 a comparison of the tumor sequence with the normal germline Thunberg et al45 2001 107 45 55 sequence. Various databases are available to enable this com- Krober et al46 2001 300 46 54 parison, but these differ in the number of polymorphic sites Oscier et al 2001 204 60 40 that are recognized. A further problem is that the incidence (unpublished) of polymorphisms differs among the VH gene families and

Leukemia Configuration of immunoglobulin genes in B cell CLL MJS Dyer and DG Oscier 976 ted VH genes, whereas all cases with mutated VH genes had VH gene mutations, then it would be difficult to reconcile Ͻ30% CD38 expression. Subsequent studies have confirmed these data with the concept that one subset of CLL has a better

the association between unmutated VH genes and high CD38 prognosis primarily because it is a disease of a post germinal expression but discordant results (CD38-positive in mutated center B cell. No studies have yet shown the prognostic value

cases or CD38-negative in unmutated cases) have been found of VH gene status in patients in whom serum markers such as in 28–43% of patients.45,46 In addition, CD38 expression may ␤-2 microglobulin or soluble CD23 have been measured. vary in the course of the disease. In a sequential study of 41 patients, 10 showed changes in CD38 expression.46 In the majority of these, a rise in CD38 expression followed chemo- Biased VH gene usage in CLL therapy suggesting that CD38-positive cells may be more

resistant to treatment. CD38 expression alone cannot therefore The germline repertoire comprises 52 VH gene segments sub- 55 be used as a surrogate marker for VH gene status. divided into seven families, six JH and 27 DH segments. Whether other cell surface proteins or combinations of pro- Some of these VDJH segments are utilized more frequently teins, indicative of B cell passage through the germinal center than others in the normal B cell repertoire. There is increasing can be used to enhance the speciﬁcity and sensitivity of a evidence that usage of the different gene segments is not ran- FACs-based assay remains to be determined. dom.56 This biased usage occurs in part before antigen exposure since it is seen in the non-productive rearrangement,57 but in part, is also due to antigen selection. A prime example of this in disease is the exclusive use of the V4–34 SPOTLIGHT Prognostic signiﬁcance of VH mutations in CLL gene in monoclonal cold agglutinins directed against the Ii In 1999, Hamblin et al40 and Damle et al43 simultaneously antigens.

reported the prognostic significance of VH gene status in CLL. Difficulties in identifying biased usage include incomplete Both groups used homology of у98% to the germ line knowledge of the normal repertoire and the influence of age-

sequence to deﬁne the absence of SHM. The median survival ing, since there are data to suggest that the VH gene repertoire of unmutated cases was 117 and 108 months, respectively. In alters with advancing age.58,59 Total knowledge of the normal our series the median survival of mutated cases was 293 repertoire is currently based on an analysis of B cell clones months, while the median survival in Damle’s series had not from small numbers of healthy individuals. Nevertheless,

been reached. These data have been corroborated in more many studies have noted that certain VH gene segments such recent studies. The median survival of unmutated cases was as V1–69, V3–23 and V4–34 are regularly used in CLL.41,42,60 77 months in a study that included 300 patients47 and 108 Widhopf and Kipps61 found that leukemic B cells which util- months in a second study of 24 cases.48 In neither study was ized 51p1, an allele of V1–69, also showed biased usage of 49 the median survival of mutated cases reached. Maloum et al DH and JH segments resulting in a long ‘conserved’ CDR3 noted that eight of 12 patients with unmutated VH genes died region. Of particular interest is the distribution of mutations from CLL-related causes compared to only two of 27 cases within the commonly used VH genes. Most V1–69 genes with mutated VH genes. remain unmutated, whereas the converse is true for the V4– The prognostic signiﬁcance of VH gene status is evident 34 gene. The possible signiﬁcance of this is discussed below. within all clinical stages of CLL. In our own study the median

survival of stage A patients with unmutated VH genes was 95 months compared to 293 months for patients with mutated The possible role of antigen selection in CLL

VH genes. Although the prognostic significance of VH gene status is unequivocal, the relative importance of VH gene A number of studies have shown that the pattern of VH status in relation to other known prognostic factors is still mutations is consistent with antigen selection in a minority of uncertain. Some studies have found CD38 expression to be a cases.43,48,60 Interpretation of these data is problematic for a 60 weak independent prognostic factor in patients of known VH number of reasons. Most studies have used the algorithm gene status, whereas others have not confirmed this find- proposed by Chang and Casali62 to define antigen selection, ing.45,48,50,51 The prognostic significance of cytogenetic abnor- but recently Lossos et al63 have suggested a different multi- malities in CLL is well documented.1 Cases with a normal kar- nomial distribution model to assess antigen selection. When yotype or deletion of chromosome 13q14 have a significantly both models were applied to 86 cases, opposite conclusions better prognosis than cases with deletions of chromosomes about antigen selection were found in eight cases. Another 11q or 17p. Preliminary data from our own group52 and from potential problem is that if a transforming event that reduces Krober et al47 have shown that deletions of 11q and 17p are susceptibility to apoptosis has occurred in leukemic cells prior

more common, but not exclusive to, cases with unmutated VH to exposure to the mutator mechanism, then the selective genes and that del 17p is an independent adverse prognostic pressure of antigen would no longer apply. Finally, until one factor in a multivariate analysis which includes clinical stage, or more antigens are discovered against which the leukemic 53 VH gene status and CD38 expression. Pettitt et al have clone is directed, then discussions on the incidence and recently shown that failure of CLL cells to upregulate importance of antigen selection in CLL remain hypothetical. expression of p21 in response to in vitro irradiation indicates p53 dysfunction due either to p53 mutation or inactivation of ATM. The same group has subsequently found that in vitro The biological significance of VH mutations resistance to purine analogues is confined to a subgroup of patients with Ͻ5% divergence from the germline sequence The clinical, morphological and genetic heterogeneity that and evidence of p53 dysfunction.54 exists within CLL is consistent with a slowly progressive neo- Whether the degree of SHM has prognostic significance plastic disorder characterized by genomic instability and in remains controversial. If this was confirmed in larger series which patients may present at different stages of the disease. using a standardized method for assessing the percentage of However, the discovery of two mutational subsets in CLL

Leukemia Configuration of immunoglobulin genes in B cell CLL MJS Dyer and DG Oscier 977 raises the issue as to whether CLL is really two diseases rather abnormalities of CSR. Interphase FISH studies using probes than one. The only reasonable explanation for CLL cases with spanning the IGH locus indicate that the frequency of such SHM, but without clonal heterogeneity, is that the final trans- translocations is genuinely low, and probably less than 2% of forming event has occurred in a cell that has been exposed to all cases of CLL. However, the frequency of CSR aberrations the mutator mechanism, presumably within a germinal center. in CLL (which would not be detected by the assay) has not Unmutated cases, by analogy, are those that have not been been determined. Laffan and Luzzatto69 documented two exposed to the mutation process, although it is at least theor- cases of B cell prolymphocytic leukemia with an unexpected ␮ etically possible that cells could traverse a germinal center BamHI site with lack of comigration of C and JH, which were without undergoing hypermutation or that the neoplastic pro- shown by bacteriophage cloning to represent an inversion cess could prevent cells from undergoing SHM. within IGH, presumably arising as an error of CSR, resulting More contentious is the nature of the unmutated cell. The in a head-to-head juxtaposition of C␮ and one of the down- phenotype of weak surface immunoglobulin and expression stream C␥ segments.69 This kind of abnormality would appear of CD23, CD27 and CD38 is not that of a naı¨ve B cell, but to be relatively rare, occurring in 4/178 cases studied by Laf- more consistent with a cell which has been exposed to anti- fan and Luzzatto. gen and perhaps rendered anergic. The failure of such cells In contrast, local abnormalities may be seen in up to 30– to undergo apoptosis may be a consequence of the neoplastic 40% of cases of CLL. Limited DNA sequencing of such process, which promotes cell survival. The most compelling rearrangements has shown that these changes represent both evidence that antigenic exposure has occurred is the finding mutations as well as insertions, duplications and deletions ␮ ′ of biased VH gene usage in cells that are unmutated. It is poss- throughout the JH-C intron, but specifically within the 5 por- ible that a putative autoantigen or exogenous infective agent tion of the S␮ region. An example of one case in which a may be T independent or may bind antibody via the frame- point mutation introduced a novel BamHI site is shown in work region (superantigen). In either case, B cell proliferation Figure 2. From restriction map analysis, these changes appear would not require entry in a germinal center. However, it to cluster to the region immediately 5′ to, and involving the should be stressed that there is no experimental evidence to 5′ region of the repetitive switch region upstream of C␮. How- support these hypotheses. ever, a comprehensive sequence analysis of such alterations Preliminary data from cDNA microarray experiments sug- in a well-characterized series of CLL is lacking. Such changes gest that all CLL cells have a common expression signature, are not specific to CLL, and are seen at lower frequency in but in addition there are differences between the two subsets other B cell malignancies and presumably reflect the action 64,65 defined by VH mutations. In particular there is up-regu- of the SHM mechanism outside the recombined VDJ gene. ␮ lation of genes responsible for signaling through the B cell The relationship of these changes within the JH-C intron to receptor in cases with unmutated VH genes. Studies investigat- VH mutations and their possible significance in terms of IGH ing the genetic, biochemical and functional differences gene expression remain to be determined. If these mutations ␮ between the two subsets are ongoing, but it is already clear extend into the enhancer within the JH-C intron, they may that VH gene mutational status is a powerful prognostic factor account in part for the low-level IGH expression seen in CLL. in CLL, which should enable new therapies to be targeted to patients with aggressive disease and avoid over-treatment of patients with benign disease. Other mutational events in CLL

BCL6 mutations: A number of other mutational events ␮ Abnormalities within the JH–C intron have been observed in CLL. Firstly, mutations within the first intron of the BCL6 gene have been seen in between 23 and Both immunophenotypic and molecular genetic studies indi- 42% of cases in three studies.70–72 Comparable somatic cate that most cases of CLL have not undergone CSR on either mutations occur in normal B cells during their transition the expressed (functional) or non-functional IGH alleles. Only through the normal germinal center reaction and are thought rare cases of CLL express Ig isotypes other than IgM.66 The to represent the action of the SHM outside the confines of the 73 ␮ possible clinical significance of CSR and its relationship to VH IG loci. As with the changes in the JH-C intron, they consist mutations have not been directly explored. Given that CSR of point mutations, which may be biallelic, deletions and occurs in normal B cells after the onset of SHM, it is likely insertions. They are clustered in the same region in which SPOTLIGHT that CLL that express IgG and other isotypes will also harbor chromosomal translocation breakpoints occur in diffuse large

VH mutations. B-NHL. Similar mutations have been found recently in a num- However, in Southern blot studies a number of investigators ber of other genes, which like BCL6 also undergo chromo-

have noticed a high frequency of abnormalities within the JH- somal translocation in mature B cells. These data therefore C␮ intron in CLL (Refs 67, 68 and MJSD, unpublished suggest that genetic regions susceptible to such apparently observations). These abnormalities fall into two separate cat- SHM-mediated mutation may be targeted for translocation.74 egories. Firstly, using a C␮ probe, no rearrangements should Chromosomal translocations involving BCL6 have not been be detected by Southern blotting following digestion with a reported in CLL, but mutations within the ﬁrst intron are com- number of restriction enzymes including HindIII, PstI, SacI mon. The relevance of these mutations in terms of BCL6

and BglII, due to the presence of restriction sites in the JH- expression is not clear. CLL express only low levels of BCL6 ␮ ␮ C intron. With these enzymes, C should retain germline protein. Moreover, the relationship of BCL6 mutations to VH conﬁguration in both normal and malignant cells. Secondly, mutations is controversial. One group, in a study of 34 cases ␮ with restriction enzymes that have no sites within the JH-C reported a close relationship, with all eight cases with BCL6 ␮ 72 intron, such as BamHI, both JH and C should be on the same mutations having VH mutations. In contrast, another group DNA fragment. The presence of an unexpected BamHI site found no clear parallel, suggesting the operation of separate may be detected by lack of comigration of the two fragments mechanisms.71 Further studies are required to resolve this and may represent either IGH chromosomal translocations or apparent discrepancy.

Leukemia Conﬁguration of immunoglobulin genes in B cell CLL MJS Dyer and DG Oscier 978 SPOTLIGHT

Figure 2 Mutations, deletions and duplications within the 5′ S␮ region in B cell malignancies. Nucleotide sequence of Wien 133 clone B511 compared with that of normal fetal liver human IGH S␮ (middle sequence, accession number X56794), and to a rearranged IGH allele cloned from a patient with ALL (bottom sequence, accession number X54713). Nucleotides in bold and marked with asterisks indicate B511 mutations compared with the normal sequence. Point mutation in B511 at nucleotide 388 created a BamHI site (highlighted). Underlined regions are duplicated sequences. B511 contained several small insertions, the largest of which, 67 bp (region I) was almost an exact duplication of the preceding 67 base pair (region P). Region P also contained several point mutations compared with the germline sequences. Some of these mutations were duplicated in region I. However, region I contained further deletions not seen in region P, suggesting that point mutational mechanisms operated both before and after duplication of the region. A second, smaller region (14 base pair) had also undergone a similar duplication and mutation process.

CD79b mutations: The B cell antigen receptor (BCR) is a IG chromosomal translocations in B-CLL multimeric complex, in which sIg is non-covalently associated with two other proteins, CD79a and CD79b. These proteins Whilst nearly all subtypes of B-NHL and the majority of cases are necessary for cell surface expression and for signal trans- of myeloma are associated with specific IG chromosomal duction.75 CD79b is also necessary for the maintenance of translocations, it is clear from both metaphase cytogenetic allelic exclusion during normal B cell development.76 In keep- analysis and from interphase FISH using probes spanning the ␣ ing with their lower level expression of sIg, CLL cells express IGH locus (ie split of differentially labeled VH and C probes), either low or absent levels of CD79b mRNA. Sequence analy- that comparable rearrangements in CLL are rare.1 Early studies sis of cDNA clones from cases of CLL with low-level suggesting that t(11;14)(q13.3;q32.3) involving IGH with the expression of CD79b showed point mutations, insertions or cyclin D1 locus was common in CLL, almost certainly deletions that were largely located in the CD79b transmem- included cases of mantle cell lymphoma in leukemic phase brane and cytoplasmic domains, the latter containing the or splenic lymphoma with villous lymphocytes (SLVL). To our ITAM motif necessary for signal transduction.77 Functional knowledge, this translocation has not been reported in bona analysis of some CD79b mutations in a Jurkat model showed fide CLL. impaired BCR signaling.78 Together, these results suggest that Why CLL should lack a common recurrent IG translocation acquired CD79b abnormalities may underlie the diminished is not clear. A comparable lack of IG translocations is seen in surface BCR display and loss of BCR signaling characteristic B cell precursor acute lymphoblastic leukemia (BCP-ALL). The of CLL. However, mutations of this gene have not been con- lack of IG translocations in these two diseases may reflect the firmed in other series of CLL and the presence of mutations in lack of activity of the intronic IGH enhancer. In B cell precur- genomic DNA has not been demonstrated.79 Instead, mRNA sors the intronic enhancer does not appear to be active,25 alternative splicing with deletion of the exon that encodes the whilst in CLL the mutations, deletions and insertions within ␮ extracellular Ig-like domain, has been proposed to account for the JH-C intron may result in loss of activity. A confounding diminished BCR signaling in CLL.79 point in CLL is that some cases with translocations involving Moreover, the expression of more than one IGH isotype in 14q32 do not in fact involve the IGH locus, but instead CLL does not appear to be dependent on the presence of involve another, as yet unidentified, more centromeric locus CD79b mutations.80 Interestingly, the CD79b promoter is within the band, all the IGH complex being translocated to methylation sensitive, not only at the usual CpG methylation the other derivative chromosome (unpublished observations). sites, but also at CC(A/T)GG sites. Methylation of the latter Whether these non-IGH 14q32 translocations involve BCL11B blocked the binding of early B cell factor (EBF), which is as has been documented recently in a subset of T cell pre- necessary for transcription. This may have implications for cursor ALL cases with t(5;14)(q31;q32) remains to be epigenetic control of gene expression.81 determined.82

Leukemia Configuration of immunoglobulin genes in B cell CLL MJS Dyer and DG Oscier 979 Nevertheless IG translocations do occur in B-CLL albeit at It is of interest that the mouse E␮-BCL2 transgenic model, low frequency, probably less than 2% of all cases. Their spec- which effectively recapitulates the t(14;18)(q32.3;q21.3) in all trum and the mechanisms underlying their origins are prob- B cells, exhibits no increase in CD5+ B cells. Other B cells ably different from those seen in other B cell malignancies. accumulate due to the loss of normal apoptotic mechanisms. The pathological consequences of these translocations are This may reflect the endogenous high-level expression of deregulated expression of the incoming oncogene due to the BCL2 in CD5+ cells, and raises the question as to whether physical juxataposition of the IG enhancers either within the the IG-BCL2 translocations involve other target genes in CLL. ␮ ′ ␣ + JH-C intron or 3 of C . In some instances in B-NHL it has However, it should be noted that CD5 B cell populations in been shown that translocation is associated with mutation and mouse and man may not be directly comparable.98 deletion of the translocated allele presumably due to the con- There are two aspects to the BCL2 translocations in CLL that tinuing action of SHM. Mutations within the amino terminus have not yet been adequately explored. Firstly, there is of BCL2, a domain that has cell-cycle regulatory functions, another gene (FVT1) immediately telomeric of BCL2, which have for example been associated with transformation of low- may also be deregulated as a consequence of some 5′ BCL2 grade disease.83,84 However, due to their rarity, the possible translocations.99 The possible contribution of this gene to the ′ link of IG translocations to VH mutational status has not been pathogenesis of CLL with 5 BCL2 translocations remains to determined in most cases. be explored. Secondly, the anatomy of the rare IGH-5′ BCL2 Although rare, IG translocations in CLL are of interest for translocations has not yet been determined completely the light they shed on the pathogenesis of CLL and other B (Figure 3). The 3′ BCL2-IGH translocations do not pose any cell malignancies. anatomical problems, but the involvement of the 5′ end of the same gene with the same region of IGH demands a chromosomal inversion or insertion of DNA from one chromosome to BCL2 (18q21.3) translocations another. The origins of the telomeric DNA on either derivative chromosome would shed some light on this process. Like the BCL2 was isolated from its direct involvement in 13q14 deletions, these translocations in CLL are likely to be t(14;18)(q32.2;q21.3), which is seen in about 80% of cases complex events and not simple ‘cut and paste’ events and of follicular B-NHL. In this disease, most of the translocation imply a distinct pathogenic mechanism. breakpoints involve the 3′ region of the gene, either within the 3′ untranslated region or in a number of other cluster regions more telomeric.85–87 Analysis of both derivative chro- BCL3 (19q13.3) translocations mosomes has indicated that the breaks may occur in a restricted period of B cell development within the germinal center.88 Like BCL2, this gene was isolated through its direct involve- These translocations result in deregulated BCL2 expression, ment in t(14;19)(q32.3;q13.3) observed in two cases of CLL.100 which through its anti-apoptotic action is thought to promote Initially, this translocation was thought to be common, but survival of cells otherwise destined to die within the germinal subsequent cytogenetic and molecular (principally Southern center. This, with the accumulation of other mutations, parti- blot) analysis has shown this is not the case. The frequency cularly involving MYC on chromosome 8q24.1, which has of the translocation has been estimated to be about 0.1% of marked synergistic activities with BCL2, allows the appear- cases of CLL.101,102 Most but not all cases with this translo- ance of the full neoplastic phenotype in the context of B-NHL. cation are CLL and most of these exhibit trisomy 12 in High-level BCL2 expression is seen in most cases of CLL addition to the t(14;19)(q32.3;q13.3). Many of the cases have and this may arise from changes in promoter methylation, been seen in younger patients with rapidly progressive and/or rather than chromosomal translocations.89 Nevertheless, some transformed disease. cases do exhibit translocations. Interestingly, unlike the fol- The structure of the translocation is of some interest as most licular B-NHL that typically involve the IGH locus and cases involve one of the S␣ regions. Only one case has been ␮ specifically the JH segments, in CLL many of these translo- reported involving S . These data would suggest that the cations are ‘variant’. These translocations are analogous to the translocation occurs as an error in CSR. However, CSR in CLL ‘variant’ MYC translocations in Burkitt lymphoma as they and specifically CSR to S␣ of the productive alleles, is rare in involve the IG light chain genes on chromosomes 2p12 (IGK) CLL and these data would therefore suggest a specific patho- or 22q12 (IGL). However, they additionally differ from typical genic mechanism. Breakpoints in BCL3 occurred in two clus- SPOTLIGHT BCL2 translocations in follicular B-NHL since they involve the ter regions immediately upstream (centromeric) of the BCL3 5′ end of the BCL2 gene.90–93 These breaks are therefore some promoter and about 15 kb further upstream. In both, the 120 kb centromeric of those within the 3′ region of the gene.94 consequences of translocation were deregulated expression of The distinct anatomy of BCL2 translocations in CLL suggests full-length BCL3, since in all instances the BCL3 open-reading a mechanism distinct from that seen in follicular B-NHL. The frame was not directly affected. Other breakpoints fell outside ′ DNA 5 of the BCL2 gene is particularly CpG rich and it has either cluster region. The VH mutational status of CLL with been suggested that it may adopt a particular configuration, t(14;19)(q32.3;q13.1) is not known. which may predispose to chromosomal translocation forma- The functions of BCL3 are of some interest, since like sev- tion.95 Interestingly, from the limited work that has been done, eral other genes such as REL and BCL10 involved in the patho- it may be that the region 5′ of BCL2 may also be subject to genesis of B cell malignancy, it regulates NF-␬B activation. hypermutational changes.96 Although translocations involving BCL3 is a member of the I␬B family of proteins that regulate the 5′ region of BCL2 are seen predominantly in CLL, similar the NF-␬B family of transcription factors.103 Unlike other I␬B events are also observed in follicular B-NHL. Between 1–2% family members, BCL3 is a nuclear protein and contains ami- of cases of CLL exhibit BCL2 translocations.93,97 It would no- and carboxy-terminal transactivation domains. BCL3 appear from sequential cytogenetic and molecular analyses, associates with homodimers of p50 or p52 NF-␬B subunits. that BCL2 translocations can be a secondary event in some of These have no defined transactivation domains and, as homo- these cases (DGO, unpublished observations). dimers, may competitively inhibit the binding of transactivat-

Leukemia Conﬁguration of immunoglobulin genes in B cell CLL MJS Dyer and DG Oscier 980 SPOTLIGHT

Figure 3 Schema depicting BCL2 chromosomal translocations in follicular B-NHL and CLL. The BCL2 gene is located on chromosome 18q21.3 and is transcribed from telomere to centromere (top panel, red boxes). There are four common recurrent breakpoints within BCL2, three falling within the 3′ region of the gene, centromeric to the coding regions and a loose cluster immediately 5′ of the gene within a large CpG island. FVT1 (http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=2531) is composed of nine exons and is located immediately telomeric of BCL2 and is transcribed in the opposite orientation (green boxes). In follicular lymphomas, the IGH breaks occur almost exclusively within ′ ′ the JH segments and become fused to one of the 3 breaks within BCL2. This is shown in the middle panel. In contrast in CLL, the 5 end of the BCL2 is often involved (bottom panel). This implies that some form of chromosomal inversion has to occur in addition to the translocation. The complete nature of such translocations has not been determined. ?? denotes that the origin of the telomere in the derivative chromosomes is not known. In follicular B-NHL, the IGH locus has nearly always undergone CSR (but see Ref. 117 for an interesting novel break in S␮), whereas the conﬁguration in CLL is not known.

ing NF-␬B dimers to consensus DNA binding (␬B) sites. BCL3 may be of primary importance in the pathogenesis of the dis- can therefore act as an anti-repressor to remove homodimers ease. Similarly, the murine homolog (Evi9) was isolated from ␬B sites, so that transactivating NF-␬B dimers can bind through its ability to induce acute myeloid leukemia in an or act as a transactivator by forming complexes with homodi- insertional retroviral mutagenesis model.108,109 Although the mers at ␬B sites or enhance homodimer binding to ␬B sites. human gene is expressed in early, multipotent hemopoietic However, BCL3 also interacts with several other proteins, sug- progenitors, the contribution of this gene to the pathogenesis gesting that the protein may have other effects outside its NF- of myeloid leukemias has yet to be assessed.110 BCL11A maps ␬B regulatory functions. Functionally, BCL3 expression was closely telomeric to REL on chromosome 2p13 and is co- able to suppress apoptosis induced by withdrawal of IL4.104 amplified with REL in most cases of amplifications of this BCL3 has recently been shown to stimulate cyclin D1 region that occur commonly in Hodgkin’s disease and in expression via activation with NF-␬B p52 homodimers.105 mediastinal and other B cell non-Hodgkin’s lymphomas, E␮-Bcl3 transgenic mice which should recapitulate CLL with although the contribution of BCL11A to these malignancies is high-level BCL3 expression due to t(14;19)(q32.3;q13.1), were not yet clear.15,111 characterized by an accumulation of mature B cells in bone Like the BCL3 translocations, BCL11A translocations marrow, lymph nodes and peritoneal cavity.106 They demon- involve switch regions but in this instance in both cases ana- strated a hyper-responsive humoral immunity as evidenced by lyzed, it was S␥2 that was involved. All three cloned breaks an increased sensitivity to BCR cross-linking, increased levels fell within 175 base pairs of each other immediately upstream of autoantibody to double-stranded DNA, and a three-fold (centromeric) of a large CpG island associated with the 5′ end increase in the number of germinal center cells in lymph of BCL11A. Like BCL3 translocations therefore, the result on nodes. However, no lymphoid neoplasms were seen in trans- the derivative chromosome 14 was a head-to-head arrange- genic animals. These data indicate that like BCL2, additional ment with IGH and BCL11A in opposite transcriptional orien- genetic events over and above the deregulated expression of tations. The consequence of the translocation was deregulated BCL3 are required for the full neoplastic phenotype to emerge. expression of BCL11A. This gene exists in three common RNA isoforms. All three were co-ordinately over-expressed in cases with the translocation. Interestingly, all cases with 112 BCL11A (2p13) translocations t(2;14)(p13;q32) exhibited germline unmutated VH genes. Although this translocation is clearly rare, the BCL11A gene Again, this gene was isolated through its direct involvement is of interest since like BCL6, it encodes a highly conserved, in another rare chromosomal translocation, t(2;14)(p13;q32.3) germinal-center specific, Kru¨ppel zinc finger transcriptional in three cases of CLL and one case of immunocytoma that repressor protein. BCL6 and BCL11A interact directly indicat- progressed to leukemic phase.15 Two of the three cases of CLL ing that they may function to control the same subset of genes occurred in young children aged 10 and 15 years. This is a in mature B cells. Whether BCL11A fulfils the functions of rare, if not unique, diagnosis.107 In two, t(2;14)(p13;q32) was BCL6 in CLL, where expression of the latter gene is relatively the sole cytogenetic abnormality, indicating that this gene low, is not known. The closely related BCL11B gene is

Leukemia Configuration of immunoglobulin genes in B cell CLL MJS Dyer and DG Oscier 981 expressed predominantly in T cells, implying tight lineage who are interested in contributing a Review for this Spotlight transcriptional control of expression of these two genes.15,82 are invited to contact the Editor-in-Chief, Dr Muller Be´rat. Whether BCL11A undergoes mutation in the same manner as BCL6 in both normal and malignant B cells is also not known. References CCND2 (12p13) translocations 1 Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bul- linger L, Dohner K, Bentz M, Lichter P. Genomic aberrations and One case of Richter’s transformation of CLL has been reported survival in chronic lymphocytic leukemia. N Engl J Med 2000; 343: 1910–1916. with a t(12;22)(p13;q12) translocation involving the IGL light 113 2 Devilee P, Cleton-Jansen A, Cornelisse CJ. Ever since Knudson. chain with the promoter of the cyclin D2 (CCND2) gene. Trends Genet 2001; 17: 569–573. Again, although this translocation appears to be rare, if not 3 Corcoran MM, Rasool O, Liu Y, Iyengar A, Grander D, Ibbotson unique in CLL, the biology of the consequence is of consider- RE, Merup M, Wu X, Brodyansky V, Gardiner AC, Juliusson G, able interest. Apart from its role in regulating the cell cycle, Chapman RM, Ivanova G, Tiller M, Gahrton G, Yankovsky N, Zab- cyclin D2 has other specific roles in B cell development and arovsky E, Oscier DG, Einhorn S. Detailed molecular delineation of 13q14.3 loss in B-cell chronic lymphocytic leukemia. Blood signaling via the BCR.114 Mice in which the cyclin D2 gene + 1998; 91: 1382–1390. has been inactivated specifically lack B cells of the CD5 lin- 4 Migliazza A, Bosch F, Komatsu H, Cayanis E, Martinotti S, Toniato + eage indicating an essential role for this gene in murine CD5 E, Guccione E, Qu X, Chien M, Murty VV, Gaidano G, Inghirami B cell development. Moreover, cyclin D2-deficient mice fail G, Zhang P, Fischer S, Kalachikov SM, Russo J, Edelman I, Efstrati- to proliferate in response to signals mediated via the BCR, adis A, Dalla-Favera R. Nucleotide sequence, transcription map, although signaling via CD40 remains intact. These and recent and mutation analysis of the 13q14 chromosomal region deleted in B-cell chronic lymphocytic leukemia. Blood 2001; 97: 2098– functional data, indicate a role for cyclin D2 in antigen- 2104. dependent B cell clonal expansion via the Rho-family guan- 5 Wolf S, Mertens D, Schaffner C, Korz C, Dohner H, Stilgenbauer S, ine-nucleotide exchange factor, Vav.115 The possible role of Lichter P. B-cell neoplasia associated gene with multiple splicing disruption of this pathway in other cases of CLL lacking trans- (BCMS): the candidate B-CLL gene on 13q14 comprises more than locations of the CCND2 gene has not been investigated. 560 kb covering all critical regions. Hum Mol Genet 2001; 10: It is of interest that all three human cyclin D genes are 1275–1285. 6 Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet involved in IG translocations in varying stages of malignant B 1999; 21: 163–167. cell development. Whether the other cyclin D proteins also 7 Lorincz MC, Groudine M. C(m)C(A/T)GG methylation: a new epi- signal via the same pathway in malignant B cells is not clear. genetic mark in mammalian DNA? Proc Natl Acad Sci USA 2001; 98: 10034–10036. 8 Gardiner AC, Corcoran MM, Oscier DG. Cytogenetic, fluor- Concluding remarks escence in situ hybridisation, and clinical evaluation of translocations with concomitant deletion at 13q14 in chronic lympho- Although abnormalities of the IG genes are not directly impli- cytic leukaemia. Genes Chromos Cancer 1997; 20: 73–81. 9 Willis TG, Dyer MJS. The role of immunoglobulin translocations cated in the pathogenesis of CLL in the same way as they are in the pathogenesis of B-cell malignancies. Blood 2000; 96: in the B-NHL, study of the configuration of these genes has 808–822. allowed fundamental insights into the pathogenesis of these 10 Kuppers R, Dalla-Favera R. Mechanisms of chromosomal translo- diseases and has allowed the development of new and power- cations in B cell lymphomas. Oncogene 2001; 20: 5580–5594. ful prognostic markers. Whether these genes and proteins can 11 Bergsagel PL, Kuehl WM. Chromosome translocations in multiple be used to develop novel tumor-specific therapies remains an myeloma. Oncogene 2001; 20: 5611–5622. 12 Hatzivassiliou G, Miller I, Takizawa J, Palanisamy N, Rao PH, Iida interesting prospect. The possibility that some ‘superantigens’ S, Tagawa S, Taniwaki M, Russo J, Neri A, Cattoretti G, Clynes R, are implicated in the pathogenesis of some subsets of CLL is Mendelsohn C, Chaganti RS, Dalla-Favera R. IRTA1 and IRTA2, fascinating. Through the use of high-throughput screening novel immunoglobulin superfamily receptors expressed in B cells methods, it should become possible to identify the antigens and involved in chromosome 1q21 abnormalities in B cell malig- involved, and perhaps design small molecules that interact nancy. Immunity 2001; 14: 277–289. specifically and induce apoptosis via the tumor-associated 13 Shaughnessy J Jr, Gabrea A, Qi Y, Brents L, Zhan F, Tian E, Sawyer J, Barlogie B, Bergsagel PL, Kuehl M. Cyclin D3 at 6p21 is dysreg- immunoglobulin either directly or via the associated ulated by recurrent chromosomal translocations to immunoglob- SPOTLIGHT 116 signaling pathway. ulin loci in multiple myeloma. Blood 2001; 98: 217–223. 14 Sonoki T, Harder L, Horsman DE, Karran L, Taniguchi I, Willis TG, Gesk S, Steinemann D, Zucca E, Schlegelberger B, Sole F, Acknowledgements Mungall AJ, Gascoyne RD, Siebert R, Dyer MJS. Cyclin D3 is a target gene of t(6;14)(p21.1;q32.3) of mature B-cell malignancies. We thank our colleagues in Bournemouth, Southampton, Sut- Blood 2001; 98: 2837–2844. ton and Leicester for their help in the preparation of this 15 Satterwhite E, Sonoki T, Willis TG, Harder L, Nowak R, Arriola EL, Liu H, Price HP, Gesk S, Steinemann D, Schlegelberger B, manuscript. We thank Drs Reiner Siebert (Institute for Human Oscier DG, Siebert R, Tucker PW, Dyer MJS. The BCL11 gene Genetics, University of Kiel, Germany) for permission to quote family: involvement of BCL11A in lymphoid malignancies. Blood unpublished data. We thank Dr Wal Zani (Amgen, UK) for his 2001; 98: 3413–3420. help in the preparation of Figure 2. 16 Welzel N, Le T, Marculescu R, Mitterbauer G, Chott A, Pott C, Kneba M, Du MQ, Kusec R, Drach J, Raderer M, Mannhalter C, Lechner K, Nadel B, Jaeger U. Templated nucleotide addition and

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