Oncogene (2001) 20, 5611 ± 5622 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Chromosome translocations in multiple myeloma

P Leif Bergsagel*,1 and W Michael Kuehl2

1Department of Medicine, Division of Hematology and Oncology, Weill Medical College of Cornell University, New York, NY, USA; 2Genetics Department, Medicine Branch, National Cancer Institute, Bethesda, Maryland, USA

Multiple myeloma (MM), a malignant tumor of kinds of terminally di€erentiated plasma cells (PC). somatically mutated, isotype-switched plasma cells Pre-germinal center (GC) PC, which can secrete IgM (PC), usually arises from a common benign PC tumor or undergo IgH switch recombination to secrete called Monoclonal Gammopathy of Undetermined Sig- another isotype of immunoglobulin, do not have ni®cance (MGUS). MM progresses within the bone somatically hypermutated Ig genes. These PC usually marrow, and then to an extramedullary stage from which are short-lived, surviving for about 3 days at their site MM cell lines are generated. The incidence of IgH of origin. In contrast, post-GC PC have undergone translocations increases with the stage of disease: 50% in repeated rounds of somatic hypermutation and antigen MGUS, 60 ± 65% in intramedullarly MM, 70 ± 80% in selection before di€erentiation to PC. Post-GC PC that extramedullary MM, and 490% in MM cell lines. have undergone IgH switch recombination to a non- Primary, simple reciprocal IgH translocations, which are IgM isotype typically migrate to the bone marrow present in both MGUS and MM, involve many partners where they interact with stromal cells as non-prolif- and provide an early immortalizing event. Four erative, long-lived PC that survive about 30 days but chromosomal partners appear to account for the majority sometimes many years (MacLennan, 1998). of primary IgH translocations: 11q13 (cyclin D1), 6p21 Multiple myeloma (MM) is a malignant, progressive (cyclin D3), 4p16 (FGFR3 and MMSET), and 16q23 (c- tumor of post-GC, long-lived PC that is closely maf). They are mediated primarily by errors in IgH associated with stromal cells, and is located at multiple switch recombination and less often by errors in somatic sites in the bone marrow (Figure 1) (Kyle and hypermutation, with the former dissociating the intronic Rajkumar, 1999; Malpas et al., 1998). MM is often and 3' enhancer(s), so that potential oncogenes can be preceded by a premalignant tumor called Monoclonal dysregulated on each derivative chromosome (e.g., Gammopathy of Uncertain Signi®cance (MGUS). In FGFR3 on der14 and MMSET on der4). Secondary contrast to MM, MGUS constitutes less than 10% of translocations, which sometimes do not involve Ig loci, mononuclear cells in the bone marrow, with the are more complex, and are not mediated by errors in B number of tumor cells remaining stable unless there cell speci®c DNA modi®cation mechanisms. They involve is progression to malignant MM. MGUS converts at a other chromosomal partners, notably 8q24 (c-myc), and rate of about 1% per year to frankly malignant MM are associated with tumor progression. Consistent with that expresses the same Ig clonotype and isotype, with MM being the malignant counterpart of a long-lived PC, at least 30 ± 50% of MM preceded by MGUS. oncogenes dysregulated by primary IgH translocations in Intramedullary MM progresses within the bone MM do not appear to confer an anti-apoptotic e€ect, but marrow as manifested by an increasing fraction of instead increase proliferation and/or inhibit di€erentia- mononuclear cells in the bone marrow being replaced tion. The fact that so many di€erent primary transform- by tumor cells. Terminally, MM can progress to an ing events give rise to tumors with the same phenotype extramedullary form that is called secondary PC suggests that there is only a single fate available for the leukemia (PCL), but sometimes primary PCL occurs transformed cell. Oncogene (2001) 20, 5611 ± 5622. without prior recognition of intramedullary MM. MM cell lines are derived from primary or secondary PCL. Keywords: primary translocation; secondary transloca- All stages of the tumor have a very low capacity for tion; MGUS; myeloma proliferation, with the DNA labeling index typically progressing from 50.5% in MGUS to 51% in early MM and rarely up to 5 ± 10% of tumor cells in the Multiple myeloma is a low proliferative tumor of most advanced stages of disease. post-germinal center plasma cells

Productive stimulation with antigen causes prolifera- Intrinsic genetic instability of lymphocytes and tion and di€erentiation of B lymphocytes into two translocations in B cell tumors

Although genomic instability is intrinsic to T and B *Correspondence: P Leif Bergsagel; lymphoid cells, it is striking that 50% of deaths from E-mail: [email protected] leukemias and lymphomas are attributable to B cell Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5612

Figure 1 Timing of primary and secondary translocations in the multi-step pathogenesis of MM. The top line is a diagram of the linear progression of plasma cell neoplasia, starting from a normal plasmablast. The benign plasma cell neoplasm MGUS progresses to malignant MM with a rate of about 1% per year. Initially this tumor is con®ned to the bone marrow (intra-medullary), but with time acquires the ability to grow in extra-medullary locations (blood, pleural ¯uid, skin, etc.). Some of these extra-medullary MM can establish immortalized cell lines in vitro. Ig translocations mediated by B cell speci®c mechanisms (somatic mutation and switch recombination) represent primary events in the transformation of the plasmablast. In MGUS it is evident that the cell has passed through a period of marked chromosomal instability, with gains and losses of whole chromosomes. It is possible that an underlying genetic instability may have contributed to the primary Ig translocation. With disease progression this chromosomal instability contributes to secondary translocations that do not appear to be mediated by B cell speci®c mechanisms. These translocations are associated with more aggressive growth of the tumor, including extra-medullary spread of the tumor

tumors, with about 80% of these derived from GC or aberrant resolution of double-stranded DNA breaks, post-GC B cells (Ries et al., 2000). A possible which occur at sites in the Ig locus that speci®cally are explanation for the predominance of GC and post- targeted by one of the three B cell speci®c DNA GC tumors is that GC B cells can be subjected to modi®cation mechanisms. The mechanism(s) by which three speci®c DNA modi®cations that generate double-stranded DNA breaks are generated in the double-stranded breaks in DNA, whereas T cells and partner chromosomes involved in translocations with pre-germinal center B cells are subjected only to VDJ an Ig locus are poorly understood. However, with rare recombination (and a limited amount of IgH switch exceptions, the partner breakpoint regions lack speci®c recombination in antigen stimulated pre-GC B cells) structural features that are thought to be targeted by (Max, 1999; Papavasiliou and Schatz, 2000). Early in the B cell speci®c mechanisms, but may be related to lymphopoiesis, B cells activate the lymphoid speci®c a variety of aberrations (e.g., ATM mutations) that VDJ recombination mechanism to form and edit in¯uence genetic stability (Vanasse et al., 1999; Welzel antigen receptors by sequential, regulated DNA et al., 2001). In any case, it is hypothesized that rearrangements of the IgH and IgL loci. Late in the double-stranded DNA breaks on both chromosomes immune response, antigen stimulates B cell to undergo must occur at a stage in normal B cell development IgH switch recombination, which occurs primarily in when these mechanisms are active. For example, most GC B cells, and somatic hypermutation, which pre-germinal center mantle cell lymphoma (MCL) appears to occur exclusively in GC B cells. Errors in tumors have a t(11;14) translocation with the IgH any of these three B cell speci®c DNA modi®cations, breakpoints occurring at or very near the sites each of which can `spill over' to a€ect non-Ig genes, targeted by VDJ recombinase. In most cases, the can result in chromosomal translocations, deletions, der(14) breakpoint occurs immediately 5' to a JH insertions, and even point mutations (Kuppers et al., segment and the der(11) breakpoint occurs immedi- 1999). ately 3' to a D segment, consistent with the paired In fact, a primary event in many kinds of B cell cleavage that occurs at these sites during normal DJ tumors is dysregulation of an oncogene that is recombination (Welzel et al., 2001). Despite ongoing juxtaposed near one of the potent Ig enhancers as a somatic hypermutation in many germinal center result of translocation to an IgH (14q32) locus or follicular cell lymphoma (FCL) tumors, most tumors somewhat less often to an IgL (k, 2p11 or l, 22q11) have a t(14;18) translocation with the location of the locus (Dalla-Favera and Gaidano, 2001). It is thought IgH breakpoints similar to those seen in MCL (Jager that most of these translocations are mediated by et al., 2000). It is hypothesized that these transloca-

Oncogene Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5613 tions must have occurred during Ig receptor formation Dysregulation of c-myc as a paradigm for secondary in pre-B cells or receptor editing in immature B cells, translocations in MM although we cannot discount the possibility that VDJ recombinase can become active in some germinal Chromosomal translocations that dysregulate c-myc by center B cells. juxtaposing it with one of the three Ig loci represent Germinal center Burkitt's lymphoma (BL) tumors an essentially invariant and apparently primary event have t(8;14) translocations that do not appear to be in human Burkitt's lymphoma and murine plasmacy- mediated by the VDJ recombinase mechanisms toma tumors (Dalla-Favera et al., 1982; Shen-Ong et (Goossens et al., 1998; Neri et al., 1988). For most al., 1982). The non-translocated c-myc allele is not endemic BL tumors, the IgH breakpoints occur within expressed, corresponding to the absence of c-myc VDJ regions or near downstream JH regions but not at expression in resting germinal center B cells and or very near the sites targeted by VDJ recombinase, terminally di€erentiated plasma cells (Kakkis et al., consistent with the hypothesis that the IgH breakpoints 1989). Despite the similarity in phenotype of human are mediated by the somatic hypermutation mechan- MM and murine plasmacytoma tumors, earlier studies ism. In contrast, for most sporadic BL tumors, the IgH identi®ed t(8;14) translocations, c-myc DNA rearran- breakpoints occur within or immediately adjacent to gements, or c-myc ampli®cation in less than 10% of IgH switch regions, suggesting that the IgH break- MM tumors and cell lines (Calasanz et al., 1997; points are mediated by the IgH switch recombination Dewald et al., 1985; Lai et al., 1995; Nishida et al., mechanism. In some di€use large cell lymphoma 1997; Sawyer et al., 1995). Recently, however, it was (DLCL), the t(3;14) translocation appears to involve determined that one c-myc allele is expressed selec- simultaneously two B cell speci®c mechanisms, an IgH tively in all twelve informative MM cell lines that have switch recombinase mediated breakpoint in the IgH polymorphic mutations that distinguish mRNA ex- locus and a somatic hypermutation mediated break- pressed from the maternal and paternal c-myc alleles point downstream of the promoter of the bcl-6 gene (Shou et al., 2000; Kuehl, 2001, unpublished). This (Pasqualucci et al., 1998; Ye et al., 1995). result suggested cis-dysregulation of c-myc in all MM cell lines, and prompted a more sensitive molecular cytogenetic analysis of MM cell lines and tumors. Primary vs secondary translocations in MM Three color FISH analyses of metaphase chromosomes show that 25 of 29 (86%) MM cell lines and 17/38 Primary translocations occur as early and perhaps (45%) advanced primary MM tumors have similar, initiating events during the pathogenesis of MM, complex karyotypic abnormalities involving the c-myc whereas secondary translocations occur as progression locus at 8q24.1. Strikingly, classical t(8;14) and t(8;22) events (Figure 1). For B cell tumors, most primary reciprocal translocations comprise only 10 and 5%, translocations are simple reciprocal translocations that respectively, of the c-myc karyotypic abnormalities juxtapose a partner chromosome (and oncogene) and that were detected. Most karyotypic abnormalities are one of the Ig enhancers, and are mediated by one of complex translocations and insertions that often are the three B cell speci®c DNA modi®cation mechanisms non-reciprocal, and frequently involve three di€erent described above. For B cell tumors in which one or chromosomes. In addition, associated deletions, inver- more of the three B cell DNA modi®cation mechan- sions, duplications, and ampli®cation are sometimes isms remain active, secondary translocations could be seen. Most of these c-myc karyotypic abnormalities mediated by one of these mechanisms. However, for would not be detected by conventional cytogenetics, normal PC and PC tumors, it is thought that all three but some would be detected by SKY analyses. The of the B cell DNA modi®cation mechanisms are karyotypic abnormalities often, but not always, inactive (with the possible exception of ongoing juxtapose c-myc and an Ig enhancer, with 9/25 somatic hypermutation in some MGUS tumors) (36%) of MM cell lines and 8/17 (47%) of primary (Bakkus et al., 1992; Bergsagel et al., 1996; Sahota et tumors having a karyotypic abnormality of c-myc that al., 1996). Therefore, unless one of these mechanisms does not involve an apparent association with an Ig could be re-activated, secondary translocations would enhancer. By interphase FISH analyses, it is reported be mediated by other kinds of recombination mechan- that c-myc is associated with the IgH locus in 3/140 isms that do not speci®cally target the Ig loci but MM tumors representing all stages, and in none of 79 could involve an Ig locus. Primary translocations MGUS tumors (Avet-Loiseau et al., 1999b). Cloned would be present in premalignant MGUS, whereas t(8;14) translocation/insertion breakpoints often do not secondary translocation are expected to be rare or occur at the IgH sites targeted by the three B cell absent in MGUS. Obviously, however, the only speci®c DNA modi®cations. In addition, there are at de®nitive way to distinguish primary from secondary least two tumors in which karyotypic abnormalities of translocations would be to document the time(s) at the c-myc locus are heterogeneous. All of these results which translocations occur during the linear pathogen- support a model for MM in which dysregulation of c- esis of individual tumors. In the absence of this myc occurs as a late, progression event that is de®nitive test, we propose other criteria that might mediated by secondary translocations that do not distinguish primary from secondary translocations in involve the three B cell speci®c DNA modi®cation MM (Table 1). mechanisms.

Oncogene Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5614 Table 1 Primary vs secondary IgH translocations Primary Secondary

Timing Early (initiation) Late (progression) B cell specific mechanism Usually No (not active in PC or tumor cells) Ig locus associated Usually Less often Present in MGUS Yes No Heterogeneity in tumor population No Sometimes Type of karyotypic abnormality Simple reciprocal translocation Usually complex*

*Complex=non-reciprocal translocation or insertion; often three di€erent chromosomes, inversion, deletion, duplication, or ampli®cation

Identi®cation of Ig translocations in MM MM cells represent translocation breakpoints into or near IgH switch regions (Bergsagel et al., 1996). The Karyotypic analyses of MM tumors using conven- t(4;14) translocation generates hybrid transcripts that tional cytogenetic methods greatly underestimated the are e€ectively detected by an RT ± PCR assay. Since incidence of IgH translocations, and rarely identi®ed there is no comparable Southern blot assay for the partner chromosome except for t(11;14) (Calasanz translocations into or near JH, D, and V regions, et al., 1997; Dewald et al., 1985; Lai et al., 1995; translocations mediated by errors in VDJ recombina- Sawyer et al., 1995). At least three problems are tion or somatic hypermutation can be distinguished responsible for the inability of conventional cytoge- only by FISH analysis or cloning. Translocation netic analyses of metaphase chromosomes to detect breakpoints of each kind have been cloned by IgH translocations in MM. First, MM tumors have a conventional methods, but also through the use of very low mitotic index even when tumors are cultured inverse PCR (Willis et al., 1997). in vitro with a variety of cytokines, including IL-6. Metaphase chromosomes can be obtained from approximately 30% of MM tumors, with this success Incidence of Ig translocations in MM cell lines, rate being directly correlated with the extent of PCL, MM and MGUS karyotypic abnormality and the stage of the tumor. Second, all MM tumors have abnormal karyotypes, Through a combination of conventional cytogenetics, and many tumors have numerous and complex SKY analyses, metaphase FISH analyses, isolation of chromosome rearrangements, including non-reciprocal molecular clones, and RT ± PCR assays (Bergsagel et rearrangements, that complicate the identi®cation of al., 1996; Chesi et al., 1997, 1998a; Gabrea et al., 1999; speci®c chromosomes. Third, the IgH locus (14q32) Shaughnessy et al., 2001; Shou et al., 2000; Kuehl, has a telomeric location, and many of the partner loci 2001, unpublished results), we have identi®ed at least have telomeric (4p16; 6p25) or subtelomeric (16q23) one IgH translocation in 36/39 (92%) MM cell lines locations. Two- or three-color FISH analyses or examined, with at least 17/39 (44%) lines having two spectral karyotypic (SKY) analyses of metaphase or more di€erent IgH translocations, and at least 5/39 chromosomes largely solve the second and third (13%) lines having three or more di€erent IgH problems, with the major exception being that translocations. In preliminary metaphase FISH ana- telomeric breakpoints (4p16; 6p25) are not detected lyses using ¯anking IgL probes, we identi®ed Igl by SKY analyses (Sawyer et al., 2000). Interphase translocations in 7/30 (23%) MM cell lines, but no Igk FISH analyses using probes that ¯ank an Ig locus to translocation in the 21 MM cell lines examined detect split signals or a probe from one end of an Ig (Martelli, 2001, unpublished results). This IgL analysis locus together with a probe from a speci®c chromo- included the three cell lines lacking an IgH transloca- somal region to detect fusion signals permit a tion, and all three had Igl translocations, so that all 39 comprehensive assessment of Ig translocations in all lines analysed have either an IgH or Igl translocation. tumor cells, including MGUS. To minimize the By interphase FISH analyses (Avet-Loiseau et al., signi®cant background problems with interphase FISH 1999b, 1999c, 2001; Nishida et al., 1997), it was assays, tumor cells have been enriched by pre-selection reported that IgH translocations are present in 36/79 (e.g., CD-138 magnetic bead selection), or by restrict- (47%) of MGUS tumors, in approximately 60% of ing analysis to cells that express cytoplasmic kappa or patients with intramedullary MM, and in 70 ± 80% of lambda IgL (Ahmann et al., 1998; Avet-Loiseau et al., patients with primary or secondary PCL. Two di€erent 1999b). IgH translocations were identi®ed in 3/40 (8%) PCL Sequential hybridization of Southern blots with a tumors, but in less than 1% of intramedullary MM comprehensive set of probes that ¯ank each kind of tumors. Using metaphase FISH analyses, we analysed switch region (m,g,a,e) eciently identi®es illegitimate 30 primary MM tumors that had been selected to have switch recombination fragments (ISRF) that hybridize an abnormal karyotype and no t(11;14) detected by with only one type of either 5' or 3' ¯anking switch conventional cytogenetics (Gabrea, 2001, unpublished probe. At least 90% of ISRF detected by this assay in results). In this analysis we identi®ed: IgH transloca-

Oncogene Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5615 tions or insertions in 18 (60%) of these tumors, Iida et al., 1997; Pratt et al., 2001; Richelda et al., including two di€erent IgH translocations or insertions 1997; Ronchetti et al., 1999; Shaughnessy et al., 2001; in six (20%) tumors; Igl translocations or insertions in Bergsagel and Kuehl, 2001, unpublished). With the ®ve (17%) tumors; no tumors with Igk translocations; exception of a few translocation breakpoints within the and 10 (33%) tumors with no Ig translocation. The JH region, most of these breakpoints were identi®ed much lower incidence of Igk vs Igl translocations is using probes designed to detect ISRF, so that the consistent with conventional cytogenetic results re- results are signi®cantly biased in identifying transloca- ported for MM in the Mitelman data base, and also tions that occur within or relatively near an IgH switch with SKY analyses of 150 patients (Mitelman et al., region. In any case, a summary of the location of these 2001; Sawyer et al., 1998, 2000). 40 cloned breakpoints within the IgH locus is provided In any case, IgH translocations are present in in Figure 3. It is clear from analysing this ®gure that approximately 50% of MGUS tumors, 60% of the four most frequent recurrent translocations (4p16, intramedullary MM tumors, 70 ± 80% of PCL tumors, 11q13 plus 6p21, and 16q23) have breakpoints that fall and 90% of MM cell lines that are derived from PCL within the JH and switch regions, areas that undergo tumors. Igl translocations are present in approximately physiologic double-strand DNA breaks induced by 17% of advanced intramedullary tumors and 23% of somatic mutation and switch recombination, respec- MM cell lines, whereas Igk translocations are rare. tively. This is consistent with their mediation by errors There is no published data for IgL translocations in in these processes, and is further supported by a more MGUS or less aggressive stages of intramedullary detailed analysis of the breakpoints. MM. Since it is not clear if all MM tumors are derived For the t(4; 14) the 5' breakpoints in the IgH locus from MGUS, and whether or not MM cell lines are (on der4, and indicated in Figure 3 as upward vertical representative of primary tumors, a de®nitive inter- line) are mostly located in Sm, with the one exception pretation of these results is not possible. However, as a being located in the Sa1 portion of a hybrid Sm/Sa1. working hypothesis, we suggest that IgH translocations The 3' breakpoints (on der14) are all located occur as a primary event in approximately 50% of signi®cantly downstream, sometimes in Sm but usually MM tumors, but that secondary IgH or IgL transloca- in Sg or Sa. This suggests a very speci®c timing for a tions can occur throughout tumor progression from translocation mediated by the switch recombination MGUS to intramedullary MM to secondary PCL to machinery. Following the introduction of double- MM cell lines (Figure 1). These results also suggest strand breaks in Sm and a downstream switch region that at least 30%, but perhaps as many as 50%, of and formation of a covalent circle from the intervening MM tumors do not have a primary IgH or an IgL region, there is a failure in the subsequent joining translocation. reaction, and the IgH ends are instead joined with other available chromosome ends. There is little if any loss of sequence from chromosome 4 during this Anatomy of IgH translocations in MM process. This di€ers from the IgH-c-myc translocations reported for murine plasmacytoma tumors, where both Whereas chromosome translocations in myeloid cells breakpoints appear to occur initially within Sm, with typically result in the formation of a fusion protein subsequent remodeling of the 3' breakpoint to involve with a novel or dysregulated function, most Ig a downstream switch region, typically Sa (Kovalchuk translocations in B cell tumors, including MM, result et al., 2001). In the t(4;14) cases we have analysed there in the dysregulated expression of oncogenes by is no evidence that remodeling has occurred. juxtaposition to Ig regulatory elements (Dalla-Favera In contrast to t(4;14) which has all breakpoints into and Gaidano, 2001). There are at least three or near switch regions, t(11;14) translocation break- important enhancer elements that regulate IgH points in two of eight cases involved the JH region, expression in B cells: the intronic enhancer (Em) consistent with these translocations being mediated by located in the intron between the JH and switch mu an error in somatic hypermutation (Figure 3). None- sequences, and two powerful 3' IgH enhancers theless, translocation breakpoints were near or within downstream of the alpha genes (Ea1 and Ea2) IgH switch regions in ®ve of eight cases. We have (Max, 1999). In a translocation with a breakpoint in cloned only one reciprocal pair of breakpoints, but this the JH region, all three enhancers remain on the unique example provides convincing evidence that der14 chromosome, so that the dysregulated oncogene translocations into IgH switch regions occur at the is on der14. In contrast, as a result of a reciprocal time of normal IgH switch recombination (Gabrea et translocation into a switch region these enhancers al., 1999). In this case the translocation is actually an become dissociated so that each derivative chromo- insertion of a portion of the IgH locus into 11q13. some has the potential to dysregulate juxtaposed Approximately 12 kb centromeric to cyclin D1 there is oncogenes (Figure 2). a deletion of 25 bp of 11q13, and an insertion of the Through a combination of strategies, we and others IgH segment from Sa1toSe, that includes the have cloned 40 IgH translocation breakpoints (but no enhancer 3' of alpha-1. By comparison of the insertion IgL translocation breakpoints) in MM cell lines and breakpoints with the productive m4e switch recombi- tumors (Bergsagel et al., 1996; Chesi et al., 1996, 1997, nation, it is evident that the inserted region is derived 1998a; Gabrea et al., 1999; Hatzivassiliou et al., 2001; from the productive allele, and represents part of the

Oncogene Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5616

Figure 2 Dysregulation of FGFR3 and MMSET/WHSC1 by t(4;14) (p16;q32) in MM. The relative location of the promoters (triangles), exons (rectangles), enhancers (stars), and switch regions (circles) involved in the t(4;14) are diagrammed. Following the t(4;14) translocation, the intronic enhancer (Em) on der4 is juxtaposed to MMSET resulting in mRNA transcripts that initiate in the Jh and Im exons, and downstream of the breakpoint on 4p16. The 3' enhancer (3'Ea) on der14 dysregulates the expression of FGFR3 and occasionally also results in reciprocal hybrid mRNA transcripts between the non-coding exons of MMSET and a constant region (g)

region that would normally have been excised, ciated deletions are related to the occurrence of the converted to a covalent circle, and deleted from the breakpoints within the fragile site (FRA16D) located in cell following switch recombination. The resultant this region (Bednarek et al., 2000; Krummel et al., orientation is 11pter. . .cen. . .myeov. . .//Se..Ea1...Sa1// 2000; Nakazawa et al., 2000; Ried et al., 2000). The ..cyclinD1. . .11qter. As might be expected, cyclin D1, breakpoints on 16q23 also emphasize another feature which is located in the normal physiological position that is common to the IgH translocations in MM, i.e., relative to the Ea1 enhancer, is over-expressed. they are dispersed over a great distance centromeric to Interestingly, however, myeov, a gene located 300 kb a dysregulated oncogene: 50 ± 100 kb from FGFR3 centromeric is not expressed, even though it is (4p16), 12 ± 330 kb from cyclin D1 (11q13); and 550 ± dysregulated by the Em enhancer that segregates to 1350 kb from c-maf (16q23). der11 in other MM cell lines with reciprocal 11q13 In striking contrast to the recurrent 4p16, 6p21, translocations involving an IgH switch region (Janssen 11q13, and 16q23 translocations, the other frequent et al., 2000). This result is consistent with the presence recurrent translocation partner, 8q24, has IgH break- of an insulator centromeric to Ea1 that restricts the points that often are located outside of the JH and IgH long distance e€ects of this enhancer to telomeric switch regions (Figure 3). In fact, most of the t(8;14) genes. translocations in MM are not identi®ed as ISRF by the Similar to the t(11;14), the t(14;16) translocation assay described above. Together with the fact that they breakpoints occur in the JH region (one case) as well are not reported in MGUS, and have been reported in as within or near IgH switch regions (four cases). sub-populations of MM cells within a given patient, Unlike 11q13 and 4p16, we have identi®ed two MM this supports the hypothesis that these are secondary cell lines (RPMI-8226; XG-6) in which there is a translocations mediated by non-B cell speci®c mechan- variant translocation, i.e., t(16;22)(q23;q11). In two isms. Other translocation breakpoints (1q21, 6p25, (OCI-My5; JJN-3) of three MM cell lines from which 20q11, 21q22, 22q12) have been cloned from only a we have cloned the breakpoints from both derivatives, single tumor, are infrequently reported in primary we ®nd evidence of large deletions of 16q23 sequences, patient samples, and have breakpoints that, like 8q24, whereas we found only very small deletions of 4p16 fall outside regions of physiologic recombination and 11q13 for the three t(4;14) and one t(11;14) (Figure 3). We suspect that most of these may also translocations in which sequences from both deriva- represent secondary genetic rearrangements associated tives were obtained. Perhaps the translocation-asso- with tumor progression.

Oncogene Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5617

11q13 6p21

Figure 3 Relative location of IgH translocation breakpoints in MM. Primary translocations (4p16, 11q13, 6p21 and 16q23) have breakpoints that fall primarily within switch regions, or less frequently JH regions that undergo somatic mutation. Translocations involving other loci have breakpoints that mostly fall within other regions of the IgH locus. The location of the J5, J6 and Cm exons, the intronic enhancer (Em) and switch mu region are diagrammed to scale, and highlighted by vertical dot-dashed lines. The location of breakpoints is indicated by a short vertical line below the horizontal line for der14 breakpoints, and above for the breakpoint on the other derivative. The lines are solid for the mu region and dashed for gamma, alpha, and epsilon regions. The numbers refer to individual tumors or cell lines. For 4p16: 1 H929, 2 JIM3, 3 KMS11, 4 OPM2, 5 PCL. 1 tumor, 6 UTMC2, 7 KHM11, 8 LB375 tumor, 9 LB1017 tumor, 10 T9283 tumor. For 11q13: 1 KMS12, 2 MM-S1, 3 SK-MM2, 4 U266, 5 FLAM76, 6 LB411 tumor, 7 KMM1 6p21, 8 Karpas 620. For 16q23: 1 ANBL6, 2 JJN3, 3 KMS11, 4 MM.1, 5 OCI-MY5. For other chromosomes: 1 KMM1 21q22 and 8q24, 2 FR4 8q24, 3 PCL.3 tumor 8q24, 4 SK-MM1 8q24, 5 SK-MM1 6p25, 6 SK-MM1 20q12, 7 132 bp insertion at 22q12, 8 FR4 1q21. For references see text

been well characterized, and it is not known if it Translocation partners results in an ampli®cation of cyclic levels of cyclin D1 mRNA expression, or in a loss of cell cycle control of 11q13 ± cyclin D1 mRNA levels and high-level tonic expression of cyclin By conventional cytogenetics and interphase FISH D1 mRNA. When the translocation breakpoint falls analyses the t(11;14)(q13;q32) is present in *15 ± 20% within a switch region, the intronic enhancer (and of MM (Avet-Loiseau et al., 1998; Fonseca et al., Ea1 if the breakpoint occurs downstream of this 1998). The breakpoints on 14q32 fall within either the enhancer) end up on der(11) where they may be JH region, or the switch regions, and are compatible associated with the ectopic expression of a gene, with being mediated by either somatic hypermutation myeov (myeloma over-expressed gene), located 360 kb or switch recombination, respectively (Figure 3). On centromeric to cyclin D1. This gene, which was 11q13, the breakpoints are dispersed over 330 kb identi®ed using DNA from a gastric carcinoma in a centromeric to cyclin D1, with no evidence of transformation assay, has no signi®cant sequence clustering in the Major Translocation Cluster (MTC) homology to other proteins and was found up- described for Mantle Cell Lymphoma (MCL) (Janssen regulated in 3/7 MM cell lines with 11q13 transloca- et al., 2000). This di€erence may re¯ect the di€erent tions (Janssen et al., 2000). mechanisms responsible for the translocations, as those in MCL are ascribed to errors in V(D)J recombina- 6p21 ± cyclin D3 tion, which may more speci®cally target the MTC. Alternatively it may re¯ect di€erences in accessibility Recently we identi®ed a translocation of 6p21 into of the bcl-1 locus at di€erent stages of B cell switch gamma sequence in a MM cell line (Shaugh- development. nessy et al., 2001). The breakpoint is located no more Normally B cells express cyclin D2 and cyclin D3, than 150 kb centromeric to cyclin D3, and is associated but not cyclin D1. As a result of the translocation with a sixfold increase level of cyclin D3 expression. cyclin D1 is juxtaposed to the powerful IgH 3' We identi®ed the t(6;14) in 1/30 MM cell lines, and in enhancer(s) on der(14), and its expression is dysregu- 6/150 patients with abnormal metaphase karyotypes. lated. The exact nature of this dysregulation has not By microarray analysis we found 3/53 patients with

Oncogene Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5618 high levels of cyclin D3 expression, and determined only one copy of MMSET and it is a likely candidate that these patients had a juxtaposition of the IgH locus to contribute to this phenotype. and cyclin D3 by interphase FISH analysis. Another FGFR3 is one of four high-anity tyrosine kinase MM tumor had a t(6;22) (p21;q11), so that cyclin D3 is receptors for the FGF family of ligands. It is not bracketed by the centromeric IgH and telomeric IgL normally expressed in plasma cells, and is ectopically breakpoints. The ®nding of dysregulation of cyclin D1 expressed as a result of the translocation. Activating in *15 ± 20% and of cyclin D3 in 5% of patients mutations of the t(4;14) translocated FGFR3 allele are highlights the importance of the D-type cyclins in MM. present in a third of the MM cell lines and in some There is one report of a t(12;22) (p13;q11) in a patients, substantiating an oncogenic role for FGFR3 lymphoma with a breakpoint in the negative regulatory in MM. Interestingly, some of the lines with t(4;14) element of the cyclin D2 promoter (Qian et al., 1999). translocation without FGFR3 mutations contained Although no expression data was shown it was activating mutations of N or K-ras, but no lines postulated to dysregulate cyclin D2. There is also a contained mutations of both FGFR3 and ras (Chesi et report of a MM tumor and a MM cell line in which al., 2001). This suggests that during tumor progression there is a t(12;14) translocation that may re¯ect activation of FGFR3 or ras may provide an equivalent dysregulation of cyclin D2 (Avet-Loiseau et al., stimulus to the cells (Figure 1). Activated FGFR3 has 1999a). Furthermore it emphasizes the importance of been shown to be an oncogene that can induce carefully studying the entire cyclin D pathway in MM transformation in ®broblasts that is inhibited by with particular focus on the inhibitors of CDK4 that dominant negative inhibitors of the ras/MAPK path- have been implicated in other tumors. way. Signaling from FGFR3 in MM has been shown to result in phosphorylation of STAT3 and MAPK, and to synergize with IL6 signals in an IL6-dependent 4p16.3 ± FGFR3 and MMSET plasmacytoma cell line. FGFR3 signaling can sub- The t(4;14) is identi®ed in approximately 15% of MM stitute for IL6 for the growth and survival of this line, tumors (Avet-Loiseau et al., 1998; Chesi et al., 1998b, and inhibition of these signals results in apoptosis 2001; Malgeri et al., 2000). The breakpoints occur in (Plowright et al., 2000). These experiments validate the telomeric region of chromosome 4 and result in a FGFR3 as an important target for experimental karyotypically silent translocation: it cannot be therapeutics in t(4;14) MM. identi®ed by either G-banding or spectral karyotype analysis. In the IgH locus the breakpoints all occur in 16q23 ± c-maf the switch region, and dissociate the intronic enhancer from the 3' enhancer. The 4p16 breakpoints fall 50 ± Translocation t(14;16) (q32;q23) is identi®ed in 5/39 100 kb centromeric to FGFR3 that becomes associated (13%) MM cell lines, and in 5 ± 10% of patients with with the 3' enhancer(s) on der14. This region includes MM (Chesi et al., 1998a; Sawyer et al., 1998, 2000). the 5' non-coding (largely) exons of MMSET/WHSC1 The ®ve cloned breakpoints on 16q23 occur over a that becomes dysregulated by juxtaposition to the IgH region 550 ± 1350 kb centromeric to c-maf, all but one intronic enhancer, resulting in formation of hybrid of them within the 800 kb intron of an oxidoreductase mRNA transcripts with the JH and I-mu exons. The gene, WWOX/FOR (Bednarek et al., 2000; Ried et hybrid transcripts provide a very speci®c and sensitive al., 2000). This region is a common fragile site, means of detecting this translocation. We have not FRA16D, that has been found to undergo frequent identi®ed any variant translocations, nor any translo- homozygous deletion in adenocarcinomas of stomach, cations into the JH region, suggesting that dysregula- lung, colon and ovary. Allelic deletions of this region tion of both MMSET and FGFR3 may be important have not been associated with inactivation of the in the pathogenesis of MM. remaining allele so that a possible role as a tumor MMSET encodes a nuclear protein with two suppressor gene is unclear. The orientation of predicted dominant forms. A short form that includes transcription of WWOX is from centromere to an HMG domain, and a long form that additionally telomere so that the translocations are not predicted includes 4 PHD-type zinc ®ngers and a SET domain. It to result in hybrid mRNA transcripts with the IgH thus shares homology to other PHD and SET domain locus, but instead the translocations appear to proteins such as ASH1 and trithorax in drosophila.It inactivate one allele. Mutations of the remaining allele also shares these features with MLL1/HRX/ALL1, the have not been identi®ed to date (Bergsagel, 2001, gene on 11q23 involved in translocations in acute unpublished results). Presumably the DNA double- leukemia, suggesting that it may play a role in the strand breaks that occur frequently in this fragile site oncogenic transformation of MM cells. This gene falls contribute to the pathogenesis of the t(14;16) with the minimally deleted region identi®ed for Wolf- translocation. Perhaps only in MM cells is the 3' Hirschorn Syndrome, a multiple malformation syn- enhancer suciently active to dysregulate c-maf drome characterized by mental retardation and devel- located at such a distance from the breakpoints, opmental defects resulting from partial deletion of the providing a possible reason for the apparent lack of short arm of one chromosome 4 (Stec et al., 1998). 16q23 translocations in other B cell tumors. Although it is felt to be a contiguous gene syndrome, it The identi®cation of t(16;22) (q23;q11) transloca- may be predicted that patients with this syndrome have tions with breakpoints telomeric to c-maf (not

Oncogene Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5619 involving WWOX) serves to delineate the targeted families. IRTA2 is upregulated in most Burkitt region of dysregulation, and indicates that inactivation lymphoma cell lines with 1q21 abnormalities. It was of one allele of WWOX by translocation is not also up-regulated in one of three MM cell lines with required for MM cell transformation by 16q23 1q21 translocations. How this contributes to the translocation. C-maf is the cellular homologue of v- pathogenesis of MM remains to be determined. The maf, the transforming gene in the avian maf retrovirus. location of the t(1;14) (q21;q32) breakpoint far down- It is a basic zipper transcription factor that can stream of the Sa region suggests that this likely heterodimerize with jun and fos and small maf proteins. represents a secondary translocation, at least in this In lymphoid cells it has been characterized as a T cell tumor cell line. From SKY analyses of 150 tumors, the transcription factor expressed speci®cally in TH2 incidence of the t(1;14) (q21;q32) translocation is about lymphocytes that controls the expression of IL-4 (Ho 1 ± 2% of advanced intramedullary MM tumors et al., 1996). It is expressed at a high level in MM cells (Sawyer et al., 1998, 2000). with a 16q23 translocation, and at a low to undetectable level in the other MM cell lines. How c- Other partner loci maf contributes to the pathogenesis of MM tumors with t(14;16) remains to be determined. A few other loci have been reported as infrequent recurrent 14q32 translocation partners in MM (Avet- Loiseau et al., 1999a, 2001; Bergsagel et al., 1996; 8q24 (c-myc) Nishida et al., 1997; Rao et al., 1998; Sawyer et al., Translocations of 8q24 in MM are complex, and 1998, 2000). Mostly these breakpoints have not been appear to be late events involved in the progression of cloned, nor the dysregulated genes characterized. These the disease (Shou et al., 2000). This is in surprising include 1q10 ± 12, 2p23 (?N-myc), 3q21, 4q22 ± 33, 9p13 contrast to their role as a primary event in Burkitt's (?pax-5), 11q23, 20q12, 21q22 and 22q12. Many other lymphoma and mouse plasmacytoma. It serves to partner loci have been identi®ed only once, but highlight the importance of c-myc in MM, and suggests breakpoints involving these loci have not been that in early stages of MM there may be trans factors characterized either by FISH mapping or by molecular present only in the bone marrow microenvironment cloning. that serve to up-regulate c-myc. With disease progres- sion, c-myc dysregulation by chromosome transloca- tion may abrogate the requirement for these trans Concluding thoughts and questions factors and allow for increased and/or extra-medullary growth of the MM cells. Incidence of Ig translocations in plasma cell tumors As summarized above, at least one IgH translocation 6p25 ± MUM1/IRF-4 is present in most MM tumors, and the incidence A t(6;14) (p25;q32) translocation breakpoint was increases with the stage of the tumor. Moreover, the cloned from a MM cell line and found to occur incidence of multiple independent IgH translocations immediately downstream of the IRF-4 gene, a member is similarly correlated with the stage of the tumor, of the interferon regulatory factor family active in the consistent with an accumulation of secondary IgH control of B cell proliferation and di€erentiation (Iida translocations during tumor progression. The precise et al., 1997). This translocation was identi®ed by incidence of IgL translocations at di€erent stages of metaphase FISH analyses in three of 17 (18%) MM tumorigenesis remains to be determined, although it cell lines that were screened (Yoshida et al., 1999). seems clear that the incidence of Igl translocations is Although IRF-4 is normally expressed in most MM about 20% in karyotypically abnormal tumors, cell lines, the three lines with the translocation whereas the incidence of Igk translocations is much expressed much higher levels. IRF-4 was shown to be lower. The lower incidence of Igl compared to IgH transforming in ®broblasts, suggesting that it may translocations might be explained by the fact that contribute to the oncogenesis of the MM tumors. The only a minority of IgH translocations are mediated incidence of the t(6;14) (p25;q32) translocation in by errors in somatic hypermutation, with the rate of various stages of primary PC tumors, and role in somatic hypermutation being several fold lower for tumor initiation and/or progression are not clearly IgL compared to IgH (Brezinschek et al., 1998). It is established at this time. thought that the IgL variant 8q24 translocations in Burkitt's lymphoma are mediated by errors in somatic hypermutation, but for that tumor the 1q21-24 ± IRTA1 and IRTA2 incidence of Igl and Igk translocations are similar Unbalanced non-random translocations of 1q and (about 10% each) (Mitelman et al., 2001). This trisomy 1q21 ± 31 were identi®ed in 20 ± 31% of MM contrasts to the markedly di€erent incidence of Igl (Hatzivassiliou et al., 2001). A breakpoint from this vs Igk translocations in MM, a result that remains to region was cloned from the FR4 MM cell line, and be explained. As noted above, at least 30%, but resulted in the identi®cation of two novel cell surface possibly as many as 50%, of MM tumors have no receptors homologous to the Fc and inhibitory primary Ig translocations.

Oncogene Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5620 Primary vs secondary translocations tion shows that translocations involving an IgH switch region can result in dysregulation of putative onco- Apart from an involvement of VDJ recombination, genes on both derivative chromosomes, each under IgH switch recombination, and somatic hypermutation control of a di€erent enhancer. However, it is also in generating breaks in one of the Ig loci, we have possible that a single Ig enhancer, which may possess virtually no understanding regarding the selection of at least some of the properties of a locus control region the partner loci involved in primary Ig translocations. (LCR), can dysregulate two di€erent genes, a result Unlike many kinds of B cell tumors, all three B cell that has been well documented for the LCR in the beta speci®c DNA modi®cation processes that generate globin locus (Bulger and Groudine, 1999; Dillon et al., double-stranded breaks in DNA are inactive in PC 1997; Max, 1999). In addition, we have learned from tumors. As a result secondary translocations would studies of the globin locus that although the LCR not be expected to involve these B cell speci®c preferentially a€ects the closest gene, more distal genes mechanisms. The mechanisms that are involved in can be selectively expressed at certain stages of secondary translocations remain to be determined, but development. Thus there is no reason that a transloca- two observations may be relevant. First, MM tumors tion must dysregulate the gene that is closest to the often show extreme karyotypic abnormalities that translocation breakpoint (and therefore closest to the generally are associated with solid tumors, perhaps Ig enhancer). re¯ecting structural or regulatory abnormalities that a€ect DNA metabolism (Mitelman et al., 2001; Sawyer et al., 1998). Second, the anatomy of apparent Biological effects of translocations secondary translocations involving c-myc in MM di€er We understand little about the biological consequences from the anatomy of primary translocations, but share of oncogene dysregulation by the IgH translocations some of the features of translocations seen in that have been identi®ed. Perhaps, however, we can lymphoma tumors that occur in non-homologous end begin to think more clearly about this issue by posing joining/p53 double knock-out mice (Di®lippantonio et some questions. First, why is there an apparent al., 2000; Ferguson et al., 2000). In addition to promiscuity of translocation partners (oncogenes) understanding mechanisms, the identi®cation of sec- involved in primary IgH translocations despite the fact ondary translocations raises interesting questions, that four partners (4p16, 6p21, 11q13 and 16q23) seem including some of the following: Why are the Ig loci to account for at least half of primary IgH transloca- targeted so frequently? Are only a limited number of tions, whereas no other partner seems to be implicated non-Ig loci involved? What is the basis for dysregula- in more than 1 ± 2% of tumors? This promiscuity of tion of an oncogene for translocations that do not oncogene partners stands in marked contrast to BL involve an Ig locus? Are di€erent oncogenes dysregu- (8q24, c-myc), MCL (11q13, cyclin D1) or FCL (18q21, lated by primary and secondary translocations, or is bcl-2), but shares some similarity with DLCL. One there an overlap? possibility is that a long-lived PC has a relatively ®xed phenotype that can tolerate many di€erent kinds of Anatomy of translocations oncogene dysregulation. Second, do the primary IgH translocations impact the same pathway? In fact, this Characterization of IgH translocation breakpoints in seems unlikely since there are examples of tumors that MM underscores our limited understanding of the have IgH translocations that dysregulate two of the consequences of these translocations on gene regula- major four recurrent oncogenes, i.e., FGFR3/MM.SET tion, and prompts a number of obvious questions. and c-maf; cyclin D3 and c-maf; FGFR3/MM.SET and First, what is the distance over which an Ig enhancer cyclin D1. Third, do primary Ig translocations directly can act in MM tumors? Most of the IgH breakpoints result in promotion of proliferation, inhibition of in MM are 50 ± 300 kb centromeric to a dysregulated apoptosis, or inhibition of di€erentiation of long-lived oncogene, but for the t(14;16) translocation, the PC? Although we do not have a clear answer at this breakpoints are 550 ± 1300 kb centromeric to c-maf. time, the involvement of the cyclin D genes suggests Assuming no associated deletions in this region of that promotion of proliferation is important. None of 16q23 (our preliminary FISH mapping studies suggest the translocations seem to implicate genes that inhibit that there are no major deletions), is this extreme apoptosis, which seems consistent with the phenotype distance related to some structural feature at 16q23, or of a long-lived, non-proliferative PC. Fourth, is a would this be true more generally? In this regard, it is primary translocation sucient to cause MGUS? notable that all of these breakpoints are in a fragile Again, we do not have enough information to answer site, and also within the extremely large WWOX this question but the apparently universal occurrence transcription unit. Second, can the Ig enhancers work of aneuploidy in MGUS suggests that other events are over longer distances in highly di€erentiated PC also necessary. tumors compared to less di€erentiated B cell tumors? In conclusion, studies reported mainly during the Although this is possible, translocation breakpoints past 5 years provide convincing evidence for an have been mapped about 300 kb from c-myc in some important role of Ig translocations in the pathogenesis BL (Zeidler et al., 1994). Third, how many genes are of MM. Nonetheless, it is clear that we now have more dysregulated by a translocation? The t(4;14) transloca- questions than answers about translocations in MM.

Oncogene Chromosome translocations in multiple myeloma PL Bergsagel and WM Kuehl 5621 References

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