Mechanisms of Chromosomal Translocations in B Cell Lymphomas

Mechanisms of chromosomal translocations in B cell lymphomas

Ralf KuÈ ppers*,1,2 and Riccardo Dalla-Favera1

1Institute of Cancer Genetics, Columbia University, New York, NY 10032, USA; 2Institute for Genetics and Department of Internal Medicine I, University of Cologne, Cologne, Germany

Reciprocal chromosomal translocations involving the involved in control of the cell cycle, bcl-2 functions immunoglobulin (Ig) loci are a hallmark of most mature by inhibiting apoptosis, and c-myc is a major control B cell lymphomas and usually result in dysregulated factor for cellular growth (Dalla-Favera and Gaidano, expression of oncogenes brought under the control of the 2001). As will be discussed below, the frequent Ig enhancers. Although the precise mechanisms involved involvement of Ig loci in chromosomal translocations in the development of these translocations remains re¯ects the remodeling of Ig genes that occurs during B essentially unknown, a clear relationship has been cell development as a result of V gene segment established with the mechanisms that lead to Ig gene assembly, somatic hypermutation, and class switch remodeling, including V(D)J recombination, isotype recombination. This review will focus on the involve- switching and somatic hypermutation. The common ment of these processes in chromosomal translocations denominator of these three processes in the formation associated with tumors derived from mature B cells, of Ig-associated translocations is probably represented by including B cell non Hodgkin lymphomas and multiple the fact that each of these processes intrinsically myeloma. For a discussion of chromosomal transloca- generates double-strand DNA breaks. Since isotype tion in immature B cell tumors, which often lack an switching and somatic hypermutation occur in germinal association with Ig loci, the reader is refered to recent center (GC) B cells, the origin of a large number of B reviews (Harrison, 2000; Rowley, 1999). cell lymphomas from GC B cells is likely closely related to aberrant hypermutation and isotype switching activity in these B cells. Oncogene (2001) 20, 5580 ± 5594. Remodeling Ig genes: V(D)J recombination, somatic hypermutation and class switch recombination Keywords: chromosomal translocation; class switch recombination; germinal center; lymphomagenesis; V(D)J recombination somatic hypermutation; V(D)J recombination During B cell development in the bone marrow, B cell precursors form intact exons for the variable region of Introduction antibody molecules by recombining individual Ig gene segments. For the variable region exon of the Ig heavy Distinct types of cancers often show characteristic chain, V, D and J gene segments are joined, whereas genetic changes involved in tumorigenesis. A hallmark the V region exons of k and l light chains are each of mature B cell lymphomas are balanced chromoso- composed of V and J segments (Rajewsky, 1996; mal translocations, mostly involving the immunoglo- Tonegawa, 1983). These gene rearrangements occur in bulin (Ig) genes and a variety of partner genes (Dalla- an ordered fashion by a process called V(D)J Favera and Gaidano, 2001). Some of these are recombination (Figure 1). First, a DH segment is associated with speci®c subtypes of mature B cell rearranged to a JH segment. This is followed by a VH lymphoma. Prominent examples include the bcl-1/Ig to DHJH joining, which can be either in-frame (i.e. in translocation in mantle zone lymphoma, the bcl-2/Ig the correct reading frame for encoding antibody translocation in follicular lymphoma and the c-myc/Ig sequences) or out-of-frame. In the latter case, a second translocation in Burkitt's lymphoma. In most in- attempt can be made on the other heavy chain allele, stances, the translocated partner gene becomes tran- whereas in the ®rst case, the pre B cell can proceed scriptionally deregulated as a consequence of its with development by undergoing Ig light chain gene transposition into the Ig locus. The consequences of rearrangements. such dysregulated expression of proto-oncogenes can V(D)J recombination is initatiated when two often be inferred from the normal functions of their lymphocyte-speci®c endonucleases, the recombination protein products. For example, bcl-1 is a cyclin activating gene (RAG) 1 and 2 proteins cut the rearranging gene segments at speci®c recombination signal sequences (RSS) that ¯ank the coding sequences of the V, D and J segments (reviewed in Fugmann et *Correspondence: R KuÈ ppers, University of Cologne, Department of Internal Medicine I, LFI E4 R706, Joseph-Stelzmannstr. 9, D-50931 al., 2000). Each RSS is composed of a conserved Cologne, Germany; E-mail: [email protected] heptamer sequence and a conserved nonamer separated Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5581

Figure 1 Molecular processes modifying immunoglobulin genes. V(D)J recombination is a process by which gene segments coding for the variable region of antibody molecules are assembled. Three gene segments (V, D and J) are joined to create a heavy chain V region gene, the k and l light chain genes (not shown) are each composed of V and J segments. Heavy chain assembly occurs in two steps. In the ®rst rearrangment, a DH gene segment is rearranged to a JH segment, in the second rearrangement, a VH gene is rearrranged to a DHJH joint. In the human IgH locus on chromosome 14, there are ca. 50 functional VH,27DH and six JH gene segments. In class switch recombination, the expressed heavy chain constant region (CH) gene(s) (usually the originally expressed cm and cd genes) is (are) replaced by a downstream CH gene. The recombination process involves deletion of the DNA between repetitive DNA regions (switch regions, sm,sg and sa) upstream of the recombining CH genes. The speci®city of the antibody remains unaltered, but the eector functions of the antigen receptor are changed. Somatic hypermutation introduces point mutations and also some deletions and duplications at a very high rate speci®cally into the V region genes and ¯anking sequences (mutations are indicated by vertical lines) by a 12 or 23 bp long spacer. RAG1 and 2 cleave anity to the respective antigen are positively selected. precisely between the RSS and the coding sequences of GC B cells undergo several rounds of proliferation, the two rearranging gene segments in a concerted mutation and selection before they ®nally dierentiate fashion, yielding two blunt RSS signal ends and two into memory B cells or plasma cells. covalently sealed hairpin coding ends (Figure 2a). The The process of somatic hypermutation introduces signal ends are precisely joined, releasing the interven- mostly nucleotide substitutions into rearranged V ing DNA between the rearranging gene segments as a genes; deletions and duplications account for about DNA circle. The hairpin structures of the coding ends 5% of the mutation events (Goossens et al., 1998; are opened and the two ends are joined. During this Neuberger and Milstein, 1995). Mutations are intro- reaction, nucleotides may be removed from the coding duced at a very high rate (ca. 1073 to 1074/bp/ ends by exonucleolytic digestion, and non-germline generation) in a region about 1 ± 2 kb downstream of nucleotides (N sequences) may be added by the the transcriptional promoter of both productively as lymphocyte-speci®c terminal nucleotidyltransferase well as non-productively rearranged V genes. It (TdT), further increasing the diversity of V region appears that hypermutation is dependent on the genes (Figure 2a). This phase of the reaction is presence of the Ig enhancers and on transcription of catalyzed, in addition to the RAG proteins, by the Ig genes, and that in particular transcription enzymes that are also involved in non-homologous initiation is involved (Betz et al., 1994; Fukita et al., double-strand DNA break repair, including DNA ± PK 1998; Peters and Storb, 1996). The mechanism of (which is composed of the DNA-PK catalytic subunit somatic hypermutation is still unknown. However, it and the Ku70 and Ku80 proteins), DNA ligase IV and has recently been demonstrated that hypermutation is XRCC4 (Fugmann et al., 2000). associated with double-strand DNA breaks (Bross et al., 2000; Papavasiliou and Schatz, 2000). The most favored model suggests that an error-prone DNA Somatic hypermutation polymerase is involved in the introduction of mutations When mature B cells are activated through binding of in proximity of the DNA breaks. cognate antigen with their antigen receptors and interaction with T helper cells, they migrate into B Class switch recombination cell follicles of secondary lymphoid organs (like lymph nodes and spleen) and establish histological structures A fraction of GC B cells undergoes class switch called germinal centers (GC) (MacLennan, 1994). In recombination and thereby changes the isotype of the the GC, the B cells vigorously proliferate and activate expressed BCR, resulting in altered eector functions a somatic hypermutation mechanism which speci®cally of the antibody (Figure 1) (Liu et al., 1996; Maizels, introduces mutations into the V regions of Ig genes 1999). Through class switching, the Cm and Cd genes (Berek et al., 1991; Jacob et al., 1991; KuÈ ppers et al., that are originally expressed by a naive B cell are 1993) (Figure 1). Through the acquisition of somatic subsequently replaced by the Cg,Ca or Ce genes. In mutations, antibody variants are generated, and cells the human, there are nine functional IgH constant expressing a B cell receptor (BCR) with increased region genes (1 Cm,1Cd,4Cg,2Ca and 1 Ce), located

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5582

Figure 2 V(D)J recombination and class switching (a) In V(D)J recombination, the RAG1 and RAG2 enzymes cleave the DNA at conserved recombination signal sequences (RSS) ¯anking the rearranging gene segments (here shown for a DH and JH segment). At the coding joints, hairpin structures are generated, whereas the signal joints are blunt end ligated. Upon opening of the hairpins, nucleotides may be removed from the DNA ends, or non-germline encoded nucleotides may be added before the gene segments are joined. (b) In class switch recombination, DNA is cleaved within repetitive switch regions upstream of the two recombining CH genes. The 5' part of the upstream switch region is joined to the 3' part of the downstream switch region, thereby replacing the originally expressed CH gene by a downstream CH gene. The excised DNA is circularised to a switch circle and later lost from the cell

downstream of the V, D and J gene cluster. Upstream both the switch m (sm) region and into a switch region of each CH gene, there are repetitive DNA sequences, associated with one of the downstream CH genes. The the switch regions (Figure 2b). During class switch DNA fragment between the switch regions is removed recombination, DNA strand breaks are introduced into from the chromosome and the switch regions are

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5583 joined, such that a new CH gene is placed downstream lymphoma, where the tumor cells appear to derive of the V region exon (Figure 2b). Although B cells from a population of preapoptotic GC B cells that carry only one functional VH gene rearrangement, class often acquired destructive mutations preventing ex- switching often takes place on both IgH alleles (Irsch pression of a BCR (Kanzler et al., 1996; KuÈ ppers and et al., 1994). The putative `class switch recombinase' is Rajewsky, 1998). still unknown, but the DNA ± PK complex, which is involved in V(D)J recombination and non-homologous double-strand DNA break repair (see above) is also Chromosomal translocations involving V(D)J needed for class switch recombination (Maizels, 1999). recombination

Several recurrent chromosomal translocations invol- Chromosomal translocations in B cells of healthy mice ving Ig loci in B cell lymphomas show structural and humans features which suggest involvement of V(D)J recombination in their generation. Paradigms of these B cells that carry chromosomal translocations invol- translocations are the t(11;14) bcl-1/IgH translocation ving the Ig loci can be found at a low frequency in in mantle zone lymphoma and the t(14;18) bcl-2/IgH normal mice and humans. For example, B cells translocation in follicular lymphoma (Tsujimoto et al., harbouring a bcl-2/IgH translocation have been 1985, 1988). Other examples include the t(1;14) detected at a frequency of about 1 in 104 to 106 in involving the bcl-9 gene in an acute lymphoblastic healthy individuals (Limpens et al., 1991; Summers et lymphoma and the t(1;14) involving the bcl-10 gene in al., 2001), and c-myc/IgH translocations occur at a mucosa-associated lymphatic tissue (MALT)-type lym- frequency of ca. 1 in 106 cells in normal mice and have phoma (Willis et al., 1998, 1999; Zhang et al., 1999). also been detected in 2% of healthy humans (Muller et While the present discussion is focussed on B cell al., 1995; Roschke et al., 1997). These ®ndings are lymphomas, it should be mentioned that V(D)J consistent with a multistep transformation process recombinase-associated translocations are not restricted during B cell lymphomagenesis, in which chromosomal to B cell lymphomas but also occur in T cell translocations involving Ig loci ± while playing a malignancies, involving the T cell receptor loci (Tycko decisive role in B cell lymphomagenesis and the type and Sklar, 1990). Indeed, since T cells use the same of lymphoma that develops from a transformed B enzymatic machinery for the assembly of T cell cell ± alone are not sucient to render a B cell receptor genes as B cells use to assemble Ig genes, malignant. In line with this view, transgenic mice most of the following discussion on the proposed overexpressing bcl-2 or c-myc in B cells develop B cell mechanisms of V(D)J recombination-associated trans- lymphomas that are monoclonal, implying that only locations in B cell malignancies also applies to rare transgene expressing cells will give rise to a chromosomal translocations in T cell tumors. malignant lymphoma, most likely after acquisition of additional transforming events (Adams et al., 1985; Translocation breakpoints resembling products of V(D)J McDonnell and Korsmeyer, 1991). recombination The proposed involvement of V(D)J recombination in Predominant targeting of translocations to bcl-2/IgH-type translocation events is mainly based on non-productively rearranged Ig alleles the observation that the translocation breakpoints are close to the RSS site of a JH gene segment or the 5' Most B cell lymphomas express a BCR, and the Ig RSS of a DHJH joint, implying that the translocation chromosomal translocations of these tumors are occurred as the cell attempted a DH to JH or VH to predominantely located on the non-functional Ig DHJH rearrangement (JaÈ ger et al., 2000; Tsujimoto et alleles. As there is no structural reason for why a al., 1985, 1988). This idea is further supported by the translocation event should target an Ig allele with a fact that the DH or JH gene segments at the break- functional Ig gene rearrangement less frequently than points often show trimming of the ends and that non- an allele with a non-productive rearrangement (dis- germline encoded nucleotides resembling N sequences cussed below), this indicates that there is strong are added at the breakpoints, two features which are selection for B lymphoma cells to produce a functional also typical for V(D)J joining (see above) (Bakhshi et BCR, as is the case for normal B cells (Lam et al., al., 1987; Cotter et al., 1990; JaÈ ger et al., 2000; 1997). Thus, it appears that even B cells that acquire a Tsujimoto et al., 1985, 1988). Moreover, analysis of chromosomal translocation still depend on BCR the reciprocal translocation breakpoints of bcl-1/ and expression for survival. Although there are some bcl-2/IgH translocations revealed that these breaks examples of B cell non Hodgkin lymphomas where involve DH segments (or in rare cases VH segments) translocation or mutation events resulted in loss of Ig (Bakhshi et al., 1987; Cotter et al., 1990; JaÈ ger et al., expression (de Jong et al., 1989), these cases may have 2000; Tsujimoto et al., 1988; Welzel et al., 2001). Since acquired some additional transforming events which the germline fragment on chromosome 14 that lies overcame the dependence on a functional BCR. A between the recombining DH and JH gene segments is notable exception to this rule is classical Hodgkin's deleted from the chromosome, the translocation most

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5584 likely happened when there were DNA breaks at both most other types of chromosomal translocations, the aDH and a JH gene (Figure 3a). The fact that the DH translocation event likely involves three DNA breaks genes in DH/bcl-1 and DH/bcl-2 joints show germline (at DH and JH on one chromosome and near the sequences at their 5' ends (and not VHDH joints) oncogene locus on the other chromosome). Due to the indeed argues against these translocations being loss of the DNA fragment between the DH and JH gene mediated through a DNA strand break in an already segments involved in the translocation, the bcl-1/and rearranged VHDHJH joint. bcl-2/IgH translocations are, formally, not completely A comparison of a large number of t(11;14) bcl-1/ balanced. IgH and t(14;18) bcl-2/IgH breakpoints revealed that in the t(11;14) of mantle zone lymphomas, the usage of J H Models for chromosomal breakage at the oncogene loci in genes is comparable to normal B cells, whereas in V(D)J-recombinase-associated translocations follicular lymphomas, the most downstream JH genes are preferentially involved, as are the most 5' DH genes While the involvement of aberrant V(D)J recombina- (JaÈ ger et al., 2000; Welzel et al., 2001). This led the tion in the generation of the DNA breaks of the Ig authors to speculate that the bcl-2/IgH translocations partners in translocations involving bcl-1, bcl-2 or the may occur during a particular developmental stage as other genes mentioned above appears clear-cut, the the cells are undergoing secondary DH ±JH recombina- origin the DNA breaks in the partner chromosomes is tions. However, it is still unclear why secondary DH ± less clear. Since convincing RSS motifs are seldom JH recombinations should be preferentially involved in found near the breakpoints in the oncogene loci, it is the generation of bcl-2/IgH translocations. unlikely that RSS-guided (i.e. V(D)J recombination- Taken together, it is likely that translocations of the like) DNA cleavage is involved. Moreover, a consider- bcl-2/IgH type into JH loci arise as a mistake of V(D)J able fraction of breakpoint junctions involving bcl-1 recombination and therefore involve RAG-mediated and bcl-2 show duplication of nucleotides at the DNA strand breaks within the Ig loci. As opposed to junction. This is suggestive of staggered DNA cleavage

Figure 3 Ig-gene remodeling processes involved in lymphomagenesis. Coding sequences of Ig genes are indicated in green. RSS sites are shown in (a) and (b) as black squares, and RSS-like sequences in non-Ig genes in (c) by grey quares. Non-Ig loci are shown in brown. In (g) bcl-6 is shown as an example of one of six known non-Ig genes (bcl-6, Fas, Pax-5, Rho/TTF, c-myc and Pim-1) that undergo somatic hypermutation. While bcl-6 and Fas accumulate somatic mutations in normal GC B cells, hypermutation of the four latter genes is restricted to diuse large cell lymphomas. In (h), bcl-6 is shown as an example of one of ®ve known mutating non-Ig genes (see Figure 6) that undergo translocations with breakpoints located in the mutation domain

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5585 (JaÈ ger et al., 2000; Welzel et al., 2001), a feature that is transposition event (which would result in insertion of incompatible with RAG-mediated DNA cleavage. the RSS-¯anked DNA into the recipient DNA and not Several models have been proposed to explain the a translocation), but by RAG-catalyzed hairpin chromosomal breaks in translocations of the bcl-2/IgH formation, the RSS-containing DNA fragment would type. One model is based on the fact that chi-like be excised again, leaving behind a chromosomal break sequences are found within the two predominant with two hairpin ends (Roth and Craig, 1998). Finally, breakpoint regions in the bcl-2 locus, the major a chromosomal translocation is generated when the breakpoint region (mbr) and the minor cluster region hairpin ends at the IgH locus are joined crosswise to (mcr), as well within the major translocation cluster the hairpin ends generated in the other chromosome (mtc) of the bcl-1 gene (Wyatt et al., 1992). Chi (Figure 4). sequences were originally described as hot spots of The two distinct models of chromosomal transloca- recombination in E. coli. In support of a potential role tions via transposase activity of the RAG proteins for Chi-like sequences in the bcl-1 and bcl-2 transloca- make clear predictions for the structures of the tions, an endogenous endonuclease activity detected in chromosomal breakpoints. While the same type of B lineage cells speci®cally cleaves within mbr (Jaeger et joints is generated at the DHJH or JH breaks on al., 1993). Furthermore, oligonucleotides containing chromosome 14, the reciprocal joints are dierent. In the Chi-like sequence bind to a 45 kD protein (bp45) the one-end transposition model, the 5' RSS site of the predominantly expressed in immature B cells (Jaeger et al., 1993, 1994). Based on these observations it was speculated that bp45 may bind to the Chi-like sequences and function as an accessory molecule for the endogenous nuclease(s) (Jaeger et al., 1993, 1994). Since Chi-like sequences are also present near DH and JH regions, 45 bp may also have a role in bringing together the bcl-2 and IgH loci, thereby facilitating their recombination (Jaeger et al., 1994). In two recent elegant studies it was shown that the RAG proteins, besides their function in V(D)J recombination, can also act as transposases (Agrawal et al., 1998; Hiom et al., 1998). These are enzymes that catalyze the excision of a DNA fragment ¯anked by speci®c recognition sequences and subsequent integration of this fragment into another DNA molecule. The integration is mediated through a transesteri®cation reaction and results in a duplication of several basepairs at the integration site of the recipient DNA (Agrawal et al., 1998; Hiom et al., 1998). In the transposition reaction catalyzed by RAG1 and 2, DNA fragments with RSS sites at their ends are integrated into another DNA molecule. Based on these features of the RAG proteins, Hiom et al. (1998) speculated that this activity might represent a source of oncogene translocations. They suggested two models for the generation of chromosomal translocations involving transposase activity of the V(D)J recombination machinery. The ®rst model proposes that a one-ended RAG-mediated cleavage at a DH or JH RSS creates a blunt-ended signal end which is then linked to DNA in another chromosome in a transpositional strand transfer (Hiom et al., 1998). This branched structure Figure 4 A model for RAG-mediated translocation via double- can be further processed to generate a hairpin end and ended transposition. An RSS-¯anked DNA fragment generated in an interchromosomal junction containing the RSS. The an attempted DH to JH joining (a) can be inserted into another reciprocal translocation is completed when the two DNA molecule through RAG-catalyzed transesteri®cation reac- hairpin structures ± one at the coding end of the D or tions of both RSS sites (b). If the intermediate structure is not H resolved in a transposition-like reaction, but by RAG-mediated JH gene segment and one in the other chromosome ± cleavage (c), the RSS-¯anked DNA fragment is cut out again with are joined. The second model proposes a two-ended short single stranded overhangs, leaving behind two hairpin ends insertion event (Hiom et al., 1998). Two blunt-ended (d). If these hairpin ends are crosswise ligated to the DH and JH RSS ends are involved in a coupled transposition coding hairpin ends (d), a chromosomal translocation is generated reaction on the two opposite DNA strands of the (e). The structure of t(11;14) (bcl-1/IgH) and t(14;18) (bcl-2/IgH) breakpoints in mantle zone lymphoma and follicular lymphoma, target DNA on another chromosome (Figure 4). If the respectively, are largely compatible with this model. Modi®ed intermediate structure is not resolved by a conventional from Roth and Craig (1998)

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5586 recombining DHJH or JH gene segment is found at the the bcl-2/IgH translocation in follicular lymphoma ± reciprocal joint, whereas in the two-end model, a DH occur in the GC microenvironment (JaÈ ger et al., 2000; (or VH) gene segment is present at that joint, with KuÈ ppers et al., 1999b; Willis and Dyer, 2000). At deletion of a large DNA fragment from chromosome present, there is only indirect evidence supporting 14 (Figure 3a and 4). In a collection of more than 40 formation of the t(14;18) in GC B cells. JaÈ ger et al. reciprocal breakpoints of bcl-2/IgH translocations and (2000) noted that some bcl-2/DH/JH joints in follicular in a small number of reciprocal bcl-1/IgH breakpoints, lymphoma carry point mutations in the DH genes. only a single example shows an RSS site on the Since DHJH joints can undergo low levels of somatic reciprocal chromosome, whereas all other breakpoints hypermutation, and since there are usually no muta- show a DH (or rarely a VH) gene segment, consistent tions at the bcl-2/JH breakpoints (presumably because with the second model (Bakhshi et al., 1987; Cotter et they are too far downstream of the bcl-2 promoter), it al., 1990; JaÈ ger et al., 2000; Meijerink et al., 1995; was speculated that the cells had acquired somatic Tsujimoto et al., 1988). Thus, if the breaks in the bcl-2 mutations in their DHJH joints in the GC prior to the or bcl-1 loci are indeed generated by a transposase translocation event. The idea that bcl-2 translocations activity of the RAG proteins, it occurs through a two- may occasionally take place in mature B cells in the ended reaction in the vast majority of cases. GC is also supported by an analysis of rare Ig-de®cient In summary, there are currently two models to follicular lymphomas, many of which carry only one explain DNA cleavage at the oncogene loci involved in rearranged IgH allele, the one with a bcl-2/IgH V(D)J recombination-associated translocations into Ig translocation (de Jong et al., 1989). Follicular loci, a model proposing two-ended transposase activity lymphoma is a malignancy of GC B cells, which of the RAG enzymes, and a model suggesting presumably express a functional BCR when they are involvement of another endonuclease cutting at chi- antigen-activated and driven into a GC reaction. Thus, like sequences in the breakpoint regions. Since the the bcl-2/IgH translocations of Ig-de®cient follicular structures of the chromosomal breakpoints in bcl-1 and lymphomas may have occurred after the cell became a bcl-2 translocations are compatible with both scenarios, GC B cell, perhaps by joining of bcl-2 to a downstream additional studies are needed to determine whether one JH element on the rearranged IgH allele, with of the proposed pathways indeed mediates the inactivation or deletion of the originally expressed translocations. VHDHJH joint. Arguing against a role for RAG expression in GC B cells in the generation of chromosomal translocations, Detection of T nucleotides at bcl-1/ and bcl-2/IgH however, are recent experiments in the mouse indicat- breakpoints ing that the RAG proteins are not reexpressed in the The models discussed above oer straighforward GC and that the rearranging B cells in murine spleen pathways for the generation of translocations of the are immature B cells that have immigrated from the bcl-2/IgH type. However, two recent studies suggest bone marrow and yet still express RAG proteins that the molecular events associated with these (Gartner et al., 2000; Yu et al., 1999). Indeed, Ig- translocations are more complex. JaÈ ger et al. (2000) negative (and hence presumably immature) B cells showed that a considerable fraction of bcl-1/ and bcl-2/ expressing RAG mRNA may also be found in human IgH breakpoints contain templated nucleotide inser- peripheral blood (Mere et al., 2000). Moreover, many tions, designated T nucleotides (Welzel et al., 2001). RAG-expressing cells are present outside GCs in These T nucleotides consist of short copies of DH,JH human tonsils (Girschick et al., 2001; Mere et al., or mbr sequences surrounding the breakpoints, and 1998). Hence, V(D)J recombination-associated and occur as direct as well as inverted copies. The copies thus RAG-dependent translocations may occur in are often not directly adjacent to the donor sequences secondary lymphoid organs although outside the GC. and may harbor mutations, suggesting that they are generated by an unusual process of error-prone short- Other V(D)J recombinase-mediated recombination or patch repair. It will be interesting to know how these T transposition events nucleotides are generated and whether they are also present in other types of chromosomal translocations. While the translocations discussed above involve RAG-mediated DNA cleavage at DHJH joints or JH genes and result in juxtaposition of oncogenes to Ig RAG-mediated translocation events in the GC? loci, there is also indication that the V(D)J recombi- Several recent studies suggested that V(D)J recombina- nation machinery may erroneously target genes with- tion may not be restricted to immature B cells in the out an involvement of Ig or TCR loci. Illegitimate bone marrow but may also take place in GC B cells, V(D)J recombinase activity has been implicated in allowing these cells to revise their antigen receptor by large deletions aecting the hprt locus in T cells performing novel rearrangements of their heavy or (Fuscoe et al., 1991), the transcription factor TAL1 light chain genes (Han et al., 1997; Mere et al., 1998; (Aplan et al., 1990; Brown et al., 1990) and the MTS1 Papavasiliou et al., 1997). Based on these reports, it gene (Cayuela et al., 1997). While deletions in the hprt has been speculated that (a fraction of) RAG-mediated gene have little consequences and are found at low translocations in GC B cell-derived lymphomas ± like frequency in normal T cells, the deletions in the TAL1

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5587 and MTS1 loci are likely involved in T cell of the recombining CH genes (Figure 3d). Usually, the leukemogenesis. Tal1 is a protooncogene, and dele- oncogenes involved in the switch recombination- tions disrupting 5' regulatory regions of the gene have associated translocations are translocated to the been detected in ca. 20% of T cell acute lymphoblastic der14, juxtaposing them to the IgH 3' enhancer, leukemias (T-ALL) (Aplan et al., 1990; Brown et al., which results in their dysregulated expression. How- 1990). MTS1 is a tumor suppressor gene, which is ever, since translocation events involving switch inactivated by deletion or disruption in most human regions result in translocation of the JH-Cm intron T-ALL (Cayuela et al., 1997). In each of the three enhancer to the translocation partner, this could cause genes, heptamer-like sequences are located near the dysregulation of genes also on the other derivative breakpoints of the deletions, and the breakpoints chromosome. Indeed, two examples of such a show `nibbling' of nucleotides as well as addition of combined dysregulation have been reported recently non-templated nucleotides, features which support the (Chesi et al., 1998b; Janssen et al., 2000). idea that the deletions are mediated by V(D)J Since normal class switch recombination involves recombinase activity. While each of these examples DNA cleavage at two switch regions, it is remarkable has been identi®ed in T lineage cells, there is a priori that a large number of class switch-associated no reason why similar deletion events should not translocations involve only one switch region. This occur also in B cells. holds true for all cases involving sm on the der14 A dierent type of illegitimate V(D)J recombinase chromosome; as this is the most 5' switch region, it activity was recently described in follicular lymphoma. follows that the reciprocal joints also involve sm (Chesi In two cases of the disease, the bcl-2 gene was inserted et al., 1997; Gelmann et al., 1983; Gilles et al., 2000; into the IgH locus, between a DH and JH gene Iida et al., 1997; McKeithan et al., 1997; Ohno et al., segment (Vaandrager et al., 2000). At the deletion 1993; Showe et al., 1985). In addition, the same breakpoints 5' and 3' of the bcl-2 gene on chromo- phenomenon is seen in many translocations involving some 18, cryptic RSS sites were identi®ed. Each of the the sg or sa regions. This suggests that a structure in three breakpoint junctions (bcl-2 5'/bcl-2 3', bcl-2/DH the recombination complex may favor interaction of and bcl-2/JH), showed N sequence insertions (Vaan- only one of the switch regions in the complex with the drager et al., 2000). These features strongly suggest other recombining chromosome during the transloca- that the transposition of the bcl-2 gene from tion event. Alternatively, some normal human B cells chromosome 18 into the IgH locus on chromosome may undergo a recombination event which results in 14 was RAG-mediated and happened when the cells deletion of parts of the sm region (Vaandrager et al., were undergoing a DHJH recombination. Therefore, 1998; Zhang et al., 1995), as a means of `isotype aberrant RAG-mediated recombination is likely not stabilization' to prevent class switching in IgM- only involved in the generation of reciprocal chromo- expressing B cells with a shortened sm region. As somal translocations, but also in deletions and such, some of the translocation events involving sm transposition events involving non-antigen receptor regions on both derivative chromosomes may occur genes (Figure 3b, c). during an attempted sm shortening and not during a conventional class switch recombination event. Class switch recombination is involved not only in Chromosomal translocations involving class switch reciprocal translocation events, but may also lead to recombination oncogene dysregulation by insertion events (Figure 3e). An example for this type of aberration is a case of Chromosomal breakpoints located in IgH switch multiple myeloma in which a DNA sequence deleted in regions have been detected in a large number of a switch recombination was inserted on chromosome dierent translocations, including c-myc [t(8;14)] in 11 near the cyclin D1 oncogene (Gabrea et al., 1999). sporadic Burkitt's lymphoma (Dalla-Favera et al., Since this DNA fragment harbours the IgH a1 1983; Gelmann et al., 1983; Showe et al., 1985; Taub enhancer, the insertion results in overexpression of et al., 1982), bcl-3 [t(14;19)] in B-cell chronic cyclin D1. Since such insertions are invisible by lymphocytic leukemia (McKeithan et al., 1997; Ohno standard karyotype analysis, they may be more et al., 1993), bcl-6 [t(3;14)] in diuse large cell frequent in various B cell tumors than currently lymphoma (Baron et al., 1993; Ye et al., 1993), Pax- known. 5 [t(9;14)] in lymphoplasmacytoid lymphoma (Iida et Since the detailed structure of the reciprocal al., 1996), lyt-10 [t(10;14)] in diuse large cell translocation breakpoints has been determined only lymphoma (Neri et al., 1991), MUC1 [t(1;14)] in an for a few chromosomal translocations into Ig switch extranodal lymphoma (Gilles et al., 2000), and regions, and since the molecular mechanism of class ®broblast growth factor receptor 3 [t(4;14)], c-maf switch recombination has not yet been identi®ed, the [t(14;16)] and MUM1/IRF4 [t(6;14)] in multiple events causing DNA breakage in the partner chromo- myeloma (Chesi et al., 1997, 1998a; Iida et al., somes of such translocations are largely unknown. 1997). Because of the location of the breakpoints in However, as discussed below, in some cases, somatic switch regions, these translocations likely happen as a hypermutation may be involved in causing DNA mistake of class switch recombination, when DNA breaks in the oncogenes translocated to Ig switch strand breaks are introduced into the switch regions regions.

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5588 The role of somatic hypermutation in chromosomal translocations and lymphomagenesis

Chromosomal translocations into the Ig loci: involvement of somatic hypermutation The process of somatic hypermutation not only generates nucleotide exchanges but also a signi®cant frequency of deletions and duplications (see above). As the generation of deletions/duplications is intimately associated with DNA cleavage, it was suggested that somatic hypermutation is accompanied by DNA strand breaks (Goossens et al., 1998). This model was indeed veri®ed by two recent studies demonstrating DNA double strand breaks in the V region genes of mutating B cells (Bross et al., 2000; Papavasiliou and Schatz, 2000). Since such breaks are potentially recombinogenic, somatic hypermutation may be involved directly in the generation of chromosomal translocations in B cell lymphomas (Figure 3f). An inspection of published translocation breakpoints indeed revealed several examples that suggest their generation as byproducts of somatic hypermutation (Figure 5) (Goossens et al., 1998; KuÈ ppers et al., 1999a). In Burkitt's lymphoma, the breakpoint of c-myc/IgH or /IgL translocations often lies within rearranged V genes or the J introns downstream of the rearranged V genes, i.e. throughout the region that is targeted by the hypermutation machinery (Cario et al., 2000; Denny et al., 1985; Haluska et al., 1986; Hartl and Lipp, 1987; Henglein et al., 1989; Kato et al., 1991; Klobeck et al., 1987; Neri et al., 1988). Moreover, in each of these cases, somatic mutations were identi®ed within the rearranged V genes, indicating that the loci were indeed targeted by somatic hypermutation. Importantly, RSS sites were not found near the breakpoints in these cases, arguing against involvement of the RAG enzymes. Further strong support for the notion that chromosome translocations can be generated during the course of the GC reaction is based on sequence analysis of the reciprocal translocation breakpoints which uncovered Figure 5 Translocation breakpoints suggesting an involvement somatic mutations in the 5' sequences of the V region of somatic hypermutation. Several examples of translocation genes (Cario et al., 2000; Henglein et al., 1989; Kato et breakpoint within or adjacent to rearranged V genes are shown. In all examples, somatic mutations were described in the al., 1991; Neri et al., 1988) (Figure 5). Since this part of rearranged Ig genes. The breakpoints are not associated with the Ig locus is separated by the translocation event from RSS sites and are distributed throughout the region that is the Ig enhancers, which are mandatory for hypermuta- targeted by somatic hypermutation. A question mark indicates tion (Betz et al., 1994; Goyenechea and Milstein, 1996), that the respective region has not been sequenced. Arrows indicate the position of the translocation breakpoints. Stars the translocations are likely to have occurred after indicate presence of somatic mutations. The location of the hypermutation of the rearranged V gene, i.e. after onset enhancers is exemplarily shown only in the ®rst example for the k of the GC reaction. In addition to c-myc/Ig transloca- locus. In cell line B593, the breakpoint is in the intron between tions in Burkitt's lymphoma, some translocations of the the leader and V segment exons. In cell line Ag876, a somatically mutated, non-productive V 2 gene rearrangement (V 2 ± 5/D 6± bcl-6 gene in diuse large cell lymphomas and the IgG Fc H H H 13/JH4) is present on the 8q- chromosome (unpublished receptor IIb in a follicular lymphoma also show observation). References for the lines and cases: KOBK101 (Kato translocation breakpoints in somatically mutated V et al., 1991), JI (Klobeck et al., 1987), Ly91 (Henglein et al., region genes (Akasaka et al., 2000; Callanan et al., 1989), Ag876 (Neri et al., 1988), 514, 881 (Akasaka et al., 2000), 2000), consistent with an involvement of somatic B593 (Callanan et al., 2000). sBL, sporadic Burkitt's lymphoma; eBL, endemic Burkitt's lymphoma; DLCL, diuse large cell hypermutation in the formation of these translocations lymphoma, FL, follicular lymphoma (Figure 5).

However, the detection of frequent mutations in the Somatic hypermutation of non-Ig genes in GC B cells bcl-6 gene on translocated as well as normal alleles in It was originally assumed that somatic hypermutation diuse large cell lymphomas (Migliazza et al., 1995) is a process that acts only on rearranged Ig V genes. prompted an analysis of this gene in normal B cells.

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5589 These studies revealed that the gene is often mutated in associated with hypermutation in these genes may normal human GC and memory, but not naive, B cells target them for translocation events, identifying (Pasqualucci et al., 1998; Peng et al., 1999; Shen et al., hypermutation-associated DNA breaks in non-Ig genes 1998). About one third of normal human GC and as a further potential source for chromosomal breaks memory B cells carry bcl-6 mutations, with a mutation that may result in chromosomal translocations (Figure frequency ca. 50 times lower than the corresponding 3h). Thus, in bcl-6/Ig translocations involving rear- mutation load in rearranged VH genes. Besides the ranged V genes, the DNA breaks in both the Ig and speci®c association of these mutations with GC-derived the bcl-6 loci may be hypermutation-associated. B cells, the pattern of the mutations also supports their Furthermore, bcl-6 translocations into Ig switch generation through the process of somatic hypermuta- regions may be caused by a combination of hypermu- tion (Pasqualucci et al., 1998; Shen et al., 1998). tation-associated DNA breaks in bcl-6 and class Recently, the CD95 (Fas) gene has been identi®ed as a switching-associated breaks in the Ig switch regions. second example of a gene that acquires somatic Moreover, two of the lymphoma-speci®c hypermutat- mutations in the normal GC reaction (MuÈ schen et ing loci (Rho/TTF and pim-1) loci are among the al., 2000b). As in the case of bcl-6, most CD95 many non-Ig partners of bcl-6 translocations in diuse mutations cluster downstream of the promoter. How- large cell lymphomas (Akasaka et al., 2000; Yoshida et ever, some mutations were also observed in exon 9, al., 1999). Thus, somatic hypermutation may also be which encodes the death domain. Since mutations in responsible for the origin of bcl-6 translocations that exon 9 can confer resistance to CD95-induced do not involve Ig loci. apoptosis, exon 9 mutations detected in some human B cell lymphomas (Gronbaek et al., 1998; Landowski et al., 1997; MuÈ schen et al., 2000a; Takakuwa et al., Translocations with an unclear association to Ig 2001) might allow the tumor cells to escape CD95- remodeling processes and complex translocations mediated elimination by tumor-speci®c eector T cells. Thus, the hypermutation process may also promote While most translocation breakpoints in the Ig loci can lymphomagenesis by targeting regulatory and coding be attributed to either V(D)J recombination, class sequences of the bcl-6 proto-oncogene and the CD95 tumor suppressor gene, resulting in either dysregulated expression (bcl-6) or loss of function (CD95).

Aberrant somatic hypermutation of multiple genes in diffuse large cell lymphoma In a search for other genes that are targeted by somatic hypermutation, we have recently identi®ed four genes (pax-5, c-myc, Rho/TTF and pim-1) which are frequently mutated in diuse large cell lymphomas (Pasqualucci et al., 2001). The clustering of the mutations ca 1 ± 2 kb downstream of the promoters and their pattern indicates that the mutations result from somatic hypermutation. Surprisingly, these genes are neither mutated in a number of other GC-derived B cell lymphomas nor in normal GC B cells. Thus, there appears to be a speci®c malfunction of the somatic hypermutation machinery in diuse large cell lymphoma cells or their precursors. Given the relatively high frequency of mutated genes among those analysed (4/17), it is likely that the number of loci targeted in diuse large cell lymphomas is considerably higher. Figure 6 Hypermutable regions are susceptible to chromosomal Therefore, aberrant hypermutation of coding or translocations. Shown are Ig genes and bcl-6 that undergo regulatory sequences of many genes may be the basis physiological somatic hypermutation, and four genes that are for diuse large cell lymphoma pathogenesis and targets for aberrant somatic hypermutation in diuse large cell represent a novel type of genomic instability involved lymphoma. The line below each gene indicates the region targeted in tumor development. by somatic hypermutation. The arrows indicate positions of chromosomal breakpoints. For Ig, bcl-6 and c-myc, where many Intriguingly, each of the ®ve proto-oncogenes breakpoints have been described, only representative examples are showing aberrant targeting of somatic hypermutation shown. The exact position of the breakpoint involving the Rho/ (pax-5, c-myc, Rho/TTF, pim-1 and bcl-6) are also TTF gene is unknown. Noncoding exons are shown by open involved in chromosomal translocations in diuse large boxes, coding exons by black boxes. For bcl-6, not all exons are shown. The colocalisation of the mutation and breakpoint regions cell lymphomas, with the breakpoint regions of these suggests that the hypermutation-associated DNA strand breaks genes largely overlaping with their mutated regions might increase the risk for chromosomal translocations in the (Figure 6). This suggests that the DNA breaks same regions. Modi®ed from Pasqualucci et al. (2001)

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5590 switching or somatic hypermutation, some show of IgH-associated translocations involving V(D)J re- features which do not allow a clear assignment to combinase may also be caused by dierent accessibility either of these three processes. Examples of this include of the partner loci at dierent stages of B cell translocations of either c-myc, bcl-1 or bcl-2 into the development. For example, the bcl-2 gene is expressed IgL loci, where the translocation breakpoints lie 2 ± at a higher level in pro B cells (which undergo heavy 5 kb upstream of unrearranged J gene segments in the chain gene rearrangements) than in pre B cells (which k or l locus (Adachi and Tsujimoto, 1989; Cario et al., undergo light chain gene rearrangements) (Li et al., 2000; Hollis et al., 1984; Komatsu et al., 1994). In an 1993). If transcription of the bcl-2 gene renders the bcl-2 example involving the IgH locus, the IRTA-1 gene is locus more accessible for translocation events, this could translocated into the intron between the CH3and explain the preferential targeting of bcl-2 translocations transmembrane exons of the Ca1 gene (Hatzivassiliou to the heavy chain locus. et al., 2001). In none of these cases have sequences with homology to the RSS heptamer or the switch regions been identi®ed near the breakpoints, and, of course, Occurrence of chromosomal translocations as byproducts unrearranged Ig loci or CH genes do not undergo or speci®c defects of the Ig-remodeling processes? somatic hypermutation. Perhaps the open structure of the Ig loci in B lineage cells results in a somewhat The description of chromosomal translocations as increased risk for DNA damage that may in rare `mistakes' of Ig-remodeling processes leaves it open instances give rise to translocation events independent as to whether these events occur as rare byproducts of of the B cell-speci®c recombination or hypermutation the normal processes, or represent speci®c defects in machinery. the targeting of V(D)J recombination, class switching In addition to the simple reciprocal translocations or somatic hypermutation. The ®nding that bcl-2/IgH discussed above, the Ig loci can also be involved in translocations can be detected in a large fraction of more complex translocation events. Several examples normal individuals (see above) argues against an of these have been identi®ed in multiple myeloma, involvement of somatic or inherited genetic defects in including an insertion of part of the IgH locus at a the mistargeting of V(D)J recombination. In line with t(3;8) breakpoint involving c-myc (Shou et al., 2000). the view that V(D)J recombination may have some low Similar insertion events of IgH sequences have also level intrinsic in®delity in targeting is the observation been observed in diuse large cell lymphomas with that antigen-receptor trans-rearrangements (analysed t(1;3) and t(3;6) involving the bcl-6 gene (Chaganti et for T cell receptor b and g cross-rearrangements) can al., 1998). These cases underscore the genetic instability be regularly detected in healthy humans (Kirsch and of the Ig loci in B cells and the variety of aberrant Lista, 1997; Tycko et al., 1989). Likewise, the ®nding rearrangements, in which the Ig loci can be involved. that about 30% of human GC and memory B cells carry mutations of the bcl-6 gene (Pasqualucci et al., 1998; Shen et al., 1998), argues against a role for a Heavy chain predominance of translocations somatic or germline defect in targeting of the bcl-6 gene for chromosomal translocation by aberrant Most translocations involving Ig loci in B cell lympho- somatic hypermutation (Figure 7). mas are targeted to the heavy chain locus. There are several aspects of B cell biology that likely account for this phenomenon. Obviously, class switch-associated translocation can only occur on heavy chain loci. Given the frequent involvement of switch regions in various types of translocations, this is likely one of the main factors to explain the heavy chain predominance in translocation targeting. The heavy chain locus may also be at a higher risk to acquire somatic hypermutation- associated translocations, because the average mutation load in VH region genes is nearly twice as high as the mutation load in light chain genes (Klein et al., 1998b), suggesting a higher activity of the hypermutation machinery at the IgH locus. A low frequency of translocations at the Igk locus is perhaps also due to Figure 7 Targets of somatic hypermutation. Mutations in bcl-6 the frequent inactivation of non-productive V J joints and Fas (CD95) are regarded as byproducts of physiological k k somatic hypermutation as they occur in 10 ± 30% of normal GC B by rearrangements of the kappa deleting element cells. In diuse large cell lymphomas, four genes have been (Siminovitch et al., 1985). These recombinations, which identi®ed that undergo aberrant somatic hypermutation. Since silence the respective k loci by deletion of the k these genes were identi®ed among 17 genes tested, the true enhancers, inactivate both k alleles in most l-expressing number of genes targeted by aberrant hypermutation is likely B cells, as well as the non-expressed k allele in a fraction higher. Whereas mutations at the 5' end of the Fas gene ful®l several criteria of somatic hypermutation, the association between of k-expressing B cells (Klein et al., 1998a). Besides these somatic hypermutation and the mutations in the death domain of factors relating to features of the Ig loci, a predominance the Fas gene is less certain. DLCL: diuse large cell lymphoma

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5591 Table 1 Genetic aberrations in B cell tumors generated as mistakes of B cell-speci®c Ig-modifying processes Enzyme machinery Type of molecular process Examples References

V(D)J recombination (i) translocation of oncogene to Ig (D)J locus; ± bcl-1/lgH in MZL (Bakhshi et al., 1987; Cotter et al., 1990; ± bcl-2/lgH in FL JaÈ ger et al., 2000; Tsujimoto et al., 1988) (ii) deletions in oncogenes or tumor suppressor ± a(TAL 1 and MTS1 (Aplan et al., 1990; Brown et al., 1990; genes; in T cell leukemias) Cayuela et al., 1997) (iii) transposition of genes into the Ig locus ± bcl-2 in FL (Vaandrager et al., 2000) Ig class switching (i) translocation of oncogene to IgH switch ± bcl-3/lgH in B-CLL (Chesi et al., 1997; Gelmann et al., 1983; regions; ± bcl-6/lgH in DLCL Ohno et al., 1993; Showe et al., 1985; ±c-myc/lgH in BL Baron et al., 1993; Ye et al., 1993) ± FGFR/lgH in MM (ii) insertion of excised switch sequences into ± insertion into (Gabrea et al., 1999) other loci cylin D1 in MM Somatic (i) translocation of oncogene into rearranged ± c-myc/lgH in BL see Figure 4 hypermutation V genes; ± bcl-6/lgL in DLCL (ii) somatic mutations in non-lg genes; ± bcl-6, CD95/Fas in (Migliazza et al., 1995; MuÈ schen et al., 2000b; various NHL Pasqualucci et al., 1998; Shen et al., 1998; ± Pax-5, c-myc, Pasqualucci et al., 2001) Pim-1, Rho/TTF in DLCL (iii) translocation of mutating oncogene ± bcl-6, Pax-5, Pim-1, Pasqualucci et al., 2001 Rho/TTF aSo far no example in B cell lymphomas identi®ed. Abbreviations: MZL: mantle zone lymphoma, FL: follicular lymphoma, B-CLL: B cell chronic lymphocytic leukemia, MM: multiple myeloma, BL: Burkitt's lymphoma, DLCL: diuse large cell lymphoma, NHL: Non Hodgkin lymphoma

The situation may be dierent in case of genes that that frequently have switch-associated translocations are speci®cally mutated in diuse large cell lymphomas (like Burkitt's lymphoma, diuse large cell lymphoma (e.g., pim-1, Rho/TTF, c-myc and Pax-5). Since these and multiple myeloma) (Klein et al., 1998a) argues genes are not mutated at signi®cant frequency in against a GC-independent development of these normal B cells and other types of lymphomas, their lymphomas in the course of T-independent immune occurrence may be due to a genetic alteration in diuse responses. Regarding the GC speci®city of somatic large cell lymphomas or their precursors (Figure 7). hypermutation, the presence of somatic Ig gene This would be reminescent of somatically acquired mutations in seemingly GC-de®cient animals and defects in DNA mismatch repair genes which result in humans may re¯ect the presence of GC (-like) accumulation of mutations in many genes and an structures in organs other than the lymph nodes and increased risk of colorectal carcinogenesis (Kinzler and spleen. Intriguingly, both the CD19 and the CD40 Vogelstein, 1996). knock-out mice, which were originally reported as lacking GCs, show GC (-like) structures in the Peyer's patches of the intestine, and CD40-ligand de®cient The role of the GC in B cell lymphomagenesis mice develop GCs under particular activation condi- tions (Chirmule et al., 2000; Gardby and Lycke, 2000; The frequent involvement of somatic hypermutation G Cattoretti, personal communication). Hence, it is and class switch recombination in the generation of still uncertain whether somatic hypermutation can chromosomal translocations points to the GC as the occur outside the GC microenvironment. Finally, the microenvironment in which many B cell lymphomas derivation of many B cell lymphomas from GC B cells develop (KuÈ ppers et al., 1999b). But how good is the is also supported by the morphology and immunophe- evidence that these molecular processes, and hence notype of the malignant cells and the histological their aberrant activities, are restricted to the GC? It is appearance of the tumor. For example, Burkitt's known that class switch recombination can also take lymphoma cells express the marker CD77, which is place in T cell-independent immune responses that lack speci®c of GC centroblasts. Indeed, the presence of GCs and are not associated with somatic hypermuta- structures that resemble GCs in follicular lymphoma tion (Garcia de Vinuesa et al., 1999; Maizels and and the morphological resemblance of the tumor cells Bothwell, 1985). Moreover, indication that somatic to centroblasts and centrocytes argues that this tumor hypermutation may also occur outside the GC is type represents a GC B cell lymphoma par excellance provided by the detection of somatically mutated B (Fyfe et al., 1987; Isaacson, 1996). cells in a gene knock-out mouse model that lacks GCs Although it cannot be excluded that some rare and in patients with germline mutations of the CD40- chromosomal translocations involving class switching ligand gene and absence of GCs in lymph nodes or somatic hypermutation may take place outside the (Matsumoto et al., 1996; Weller et al., 2001). However, GC microenvironment, there is little doubt that these the consistent presence of a high load of somatic translocations usually occur in the GC. Hence, besides mutations in the rearranged V genes of lymphomas the vigorous proliferation of GC B cells, which may by

Oncogene Mechanisms of chromosomal translocations in B cell lymphomas RKuÈppers and R Dalla-Favera 5592 itself increase the risk for the acquisition of genetic tion and class switching may also contribute to damage, the remodeling of Ig genes in the GC through lymphomagenesis in the GC by causing other types somatic hypermutation and class switching likely plays of mutations (Table 1, Figure 3). A particularly a decisive role in the development of many B cell intriguing example of this is the aberrant targeting of lymphomas in this microenvironment (KuÈ ppers et al., non-Ig genes by the somatic hypermutation process, 1999b). In many instances, aberrant activity of these which might be a key event in the development of two processes results in the generation of balanced diuse large cell lymphoma. chromosomal translocations. Sometimes, these two processes may even cooperate. For example, as discussed above, translocations of bcl-6 into IgH switch Acknowledgments regions may involve hypermutation-associated DNA RKuÈ ppers is supported by the Deutsche Forschungsge- breaks in the bcl-6 gene and class switch recombina- meinschaft through SFB502 and the Heisenberg pro- tion-associated DNA cleavage in the switch regions. gramme. We thank Richard Baer for critically reading Besides the generation of translocations, hypermuta- the manuscript.

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