The prognostic significance of cytogenetics and molecular profiling in multiple myeloma

Cancer Genetics 204 (2011) 3e12 REVIEW The prognostic significance of cytogenetics and molecular profiling in multiple myeloma Jeffrey R. Sawyer Department of Pathology and Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR, USA Multiple myeloma (MM) is a plasma cell malignancy characterized by very complex cytogenetic and molecular genetic aberrations. In newly diagnosed symptomatic patients, the modal chromosome number is usually either hyperdiploid with multiple trisomies or hypodiploid with one of several types of immunoglobulin heavy chain (Ig) translocations. The chromosome ploidy status and Ig rearrangements are two genetic criteria that are used to help stratify patients into prognostic groups based on the findings of conventional cytogenetics and fluorescence in situ hybridization (FISH). In general, the hypodiploid group with t(4;14)(p16;q32) or t(14;16)(q32;q23) is considered a highrisk group, while the hyperdiploid patients with t(11;14)(q13;q32) are considered a better prognostic group. As the disease progresses, it becomes more proliferative and develops a number of secondary chromosome aberrations. These secondary aberrations commonly involve MYC rearrangements, del(13q), del(17p), and the deletion of 1p and/or amplification of 1q. Of the secondary aberrations, del(17p) is consistently associated with poor prognosis. All of these cytogenetic aberrations and many additional ones are now identified by means of high resolution molecular profiling. Gene expression profiling (GEP), array comparative genomic hybridization (acgh), and singlenucleotide polymorphism (SNP) arrays have been able to identify novel genetic aberration patterns that have previously gone unrecognized. With the integration of data from these profiling techniques, new subclassifications of MM have been proposed which define distinct molecular genetic subgroups. In this review, the findings from conventional cytogenetics, interphase FISH, GEP, acgh, and SNP profiles are described to provide the conceptual framework for defining the emerging molecular genetic subgroups with prognostic significance. Keywords Multiple myeloma, cytogenetics, molecular profiling, prognosis ª 2011 Elsevier Inc. All rights reserved. Multiple myeloma (MM) is a clonal bone marrow disease characterized by the neoplastic transformation of differentiated B cells. It is a heterogeneous disease both at the genetic level and in terms of clinical outcome. Recent advances in molecular cytogenetic and genomic profiling studies have provided an increased understanding of the pathogenesis of MM. These studies have also provided the rationale for the current cytogenetic risk stratifications and molecular sub-classifications now being introduced. This review will describe the genetic studies that provide the framework for the prognostic classifications of multiple myeloma. Myeloma can progress through stages that in some cases may include a pre-malignant tumor called monoclonal Received November 1, 2010; accepted November 1, 2010. E-mail address: sawyerjeffreyr@uams.edu gammopathy of undetermined significance (MGUS) (1). MGUS is a plasma cell proliferative disorder with a plasma cell content of less than 10% in the bone marrow, a monoclonal protein spike, and involves no end organ damage. Smoldering myeloma (SM), also called asymptomatic myeloma, is an intermediate entity between MGUS and active MM. SM has a stable bone marrow tumor content of 10e30%, but no osteolytic lesions, anemia, or other secondary findings characteristic of malignant MM. Clinically malignant myeloma is a disease characterized by excess bone marrow plasma cells, monoclonal protein, osteolytic bone lesions, renal impairment, anemia, and immunodeficiency (2). Finally, extramedullary MM is more aggressive and is often called secondary or primary plasma cell leukemia. The clonal evolution of genetic aberrations found in myeloma is consistent with a multistep model of disease progression that begins in MGUS and proceeds through myeloma to extramedullary disease (1). 2210-7762/$ - see front matter ª 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2010.11.002

4 J.R. Sawyer The pathogenesis of myeloma involves complex interactions between the tumor cells and the bone marrow microenvironment. These interactions are recognized as an important contributing factor in tumor progression. This is reflected in part by the fact that early in the disease, the tumor cells are stromal dependent, but as the disease becomes more aggressive, the tumor cells become stromal independent. The marrow microenvironment is made up of extracellular matrix and five types of bone marrow stromal cells, including fibroblastic stromal cells, osteoblasts, osteoclasts, vascular endothelial cells, and lymphocytes (1). The myeloma cells can interact either directly with the bone marrow stromal cells and extracellular matrix proteins or indirectly by secretion of soluble cytokines, growth factors, and adhesion molecules. It is believed that cross-talk between myeloma cells and other cells in the bone marrow microenvironment is involved in the pathogenesis of the osteolytic lesions in MM by mechanisms involving osteoblast inactivation and osteoclast activation (3). In many newly diagnosed patients, the abnormal clones have a low proliferative activity and, therefore, most of the analyzable metaphase cells are derived from normal hematopoiesis. As a result, only about 30e40% of new patients demonstrate an abnormal karyotype by conventional metaphase techniques (4e9). The detection of an abnormal metaphase karyotype is generally correlated with an elevated plasma cell labeling index and tumor burden, which is reflected by a higher mitotic rate (10). Thus, finding abnormal metaphase mitoses in a patient can be regarded, in a certain respect, as a surrogate marker for stroma-independent cells and more advanced disease (11e13). The low proliferative activity of the tumor cells early in the disease is an important limitation of conventional cytogenetics, since only dividing cells can be analyzed. Even within cells with an abnormal karyotype, some aberrations are cryptic to metaphase analysis; for example, the t(4;14)(p16;q32) translocation is a submicroscopic aberration and cannot be detected with conventional banding techniques. These limitations have been overcome in part by the use of the molecular cytogenetic techniques of fluorescence in situ hybridization (FISH), multicolor FISH (14e17), spectral karyotyping (18e20), and comparative genomic hybridization (21e23). FISH analysis of interphase nuclei (which is independent of plasma cell division) has become an important adjunct to conventional analysis. FISH is routinely used for targeted detection of specific aberrations with established clinical significance. The standard diagnostic workup now includes both interphase FISH and traditional metaphase chromosome studies. Both studies can also be done as surveillance to monitor the therapeutic response and at relapse to help direct therapy. Recent reports reinforce the importance of both conventional cytogenetics and FISH, as both appear to be independent prognosticators at diagnosis, as well as provide complementary information (24,25). The high-risk genetic features of conventional cytogenetic and interphase FISH studies outlined below are now part of risk stratification models (25). Conventional and molecular cytogenetics in myeloma Chromosome aberrations in MM are typically complex and represent a hallmark of the disease, involving many chromosomes that are altered both numerically and structurally (4e9,18e23). This complexity is reflected in a median number of eight karyotypic changes per patient in some studies (26), while in others, the use of statistical analysis indicates that most tumors have as many as 10 abnormalities at diagnosis (27). Even at diagnosis, in many cases, these clones have already undergone expansion, generating heterogeneous subclones with many translocations that are secondary progression events (26,27). In many ways, the complexity of karyotypes in MM resemble those found in solid tumors because they combine high numbers of both numerical aberrations and very complex and unstable structural aberrations. The first prognostically significant classification of MM was based on chromosome ploidy number of conventional karyotyping. Smadja et al. (28) were the first to identify the prognostic significance of the hyperdiploid versus the nonhyperdiploid karyotype. The hypodiploid group is associated with poorer overall survival, while the hyperdiploid group does better (28). The hypodiploid classification encompasses clones composed of hypodiploid, pseudodiploid, and/or neartetraploid variants, which are almost always associated with structural aberrations. The near-tetraploid karyotypes appear to be 4n duplications of cells having pseudodiploid or hypodiploid karyotypes (28e30). The hyperdiploid group shows a consistent set of trisomies and fewer structural aberrations. The hyperdiploid karyotype is characterized by trisomies of chromosomes 3,5,7,9,11,15,19, and 21, which are observedin50e60% of patients (4,5,14,18,19). The nonhyperdiploid karyotypes typically have translocations involving the immunoglobulin heavy chain (IGH ) locus at 14q32. The most frequently lost chromosomes are 13,14,16 and 22 (28e30). It should be pointed out that the generalized distinctions between the hyperdiploid and the hypodiploid groups are an oversimplification since the primary IGH translocations are also found in hyperdiploid MM at a frequency of approximately 10% (31). It is believed that primary translocations occur early in the pathogenesis of MM, whereas secondary translocations occur later and are involved in tumor progression (1). Most primary translocations are simple reciprocal translocations and juxtapose an oncogene and one of the immunoglobulin enhancers. These translocations are mediated by errors in one of three B-cell specific DNA modification mechanisms: the most common is IGH switch recombination, followed by errors in somatic hypermutation, and rarely, VDJ recombination (12). The incidence of IGH translocations is known to increase during the progression of the disease from 50% in MGUS to 90% in human myeloma cell lines (HMCL). The incidence of light-chain translocations (kappa, 2p12 or lambda, 22q11) is far less, with about 10% being found in MGUS and 20% in advanced myeloma and HMCL (12,32). The IgH (14q32) translocations found in hypodiploid MM are promiscuous and can involve many different partners. Typically, however, there are five main translocations involving 11q13 (CCND1), 6p21 (CCND3), 16q23 (MAF ), 20q12 (MAFB), and 4p16 (FGFR3 and MMSET) (32,33). Virtually all MM and MGUS tumors have cyclin D dysregulation suggesting an early and unifying pathogenetic event. The most frequent translocation in MM is the t(11;14)(q13;q32) which is found in about 15% of patients, and appears to be associated with a favorable outcome in most series and therefore, is regarded as neutral with regard to prognosis (33e35).

Cytogenetic and molecular classifications in multiple myeloma 5 The second most frequent reciprocal translocation in MM is the t(4;14)(p16;q32), which occurs in about 15% of patients (36,37). This translocation involves two protein-coding genes that are encoded on 4p16. The first is the Wolf-Hirschhorn syndrome candidate 1 gene [WHSC1, also known as multiple myeloma SET domain (MMSET )], a protein with homology to histone methyltransferases. The second is the fibroblast growth factor receptor 3 (FGFR3)- an oncogenic receptor tyrosine kinase (1). The translocation appears unbalanced in up to 25% of cases, losing the derivative chromosome 14, which is associated with the loss of FGFR3 expression (37,38). This loss apparently reflects just one of the many possible pathways for clonal evolution during disease progression. As mentioned previously, the t(4;14) (p16;q32) is cryptic to banding and only detectable by FISH or reverse-transcriptase PCR. The t(4;14) is also associated with a high prevalence of chromosome 13 monosomy and deletions, and has universally been associated with poor survival (38e41). The t(4;14)(p16;q32) is also observed in MGUS, but is present more frequently in patients with SM and active myeloma (34,41,42). Two less frequent but clinically important translocations involve the MAF genes. The t(14;16)(q32;q23) translocation juxtaposes the IGH (14q32) locus and c-maf (16q23) locus and is found in approximately 6e7% of patients with MM. The breakpoints occur in the introns of a very large gene, WWOX, which spans a fragile site (FRA16D) on chromosome 16q23 (12). This translocation is also associated with poor outcome (34,41e43). Even less frequent is the MAFB (20q12) translocation, which occurs in about 2% of patients and involves the reciprocal t(14;20)(q32;q12). The prognostic outcome is assumed to be the same as the t(14;16). It seems unlikely that any of the IGH translocations alone would be sufficient to give rise to malignant myeloma as they are often evenly distributed between MGUS, SM, and MM (43). In MM, independent IGH translocations are unusual but do occur, indicating the possible activation of complimentary pathways by multiple primary translocations (12). These translocations include combinations of CCND3 and c-maf, c-maf and FGFR3/MMSET, and FGRF3/MMSET and CCND1. These combinations of aberrations are far more likely to be found in late disease where very complex rearrangements may also involve a large number of other secondary aberrations (12,13,32,33). Secondary aberrations in the clonal evolution of MM A large number of secondary chromosomal aberrations are found during tumor progression, but four main aberrations are most often reported. These aberrations include translocations of MYC, the loss or deletion of chromosome 13, deletions and/ or amplifications of chromosome 1, and deletion of chromosome 17p13 (1,12,44). Translocations and/or amplifications of the oncogene MYC (8q24) are involved in up to 45% of patients with advanced MM. Translocations involving MYC and the Ig locus are known to be late events in tumor progression, when tumors are becoming more proliferative and less stromaldependent (12,13). The translocations that juxtapose MYC and Ig sequences in myeloma are often very complex events involving multiple chromosomes. They can involve non-reciprocal rearrangements, duplications, and amplifications, and can be mediated by secondary translocations that do not involve B-cellespecific modification mechanisms (33). Interphase FISH studies indicate that MYC rearrangements are present in 15% of myeloma tumors, and are often heterogeneous within cells of the tumor (44). The secondary rearrangements involving Ig and MYC loci show a similar prevalence in hyperdiploid and non-hyperdiploid myeloma, as do the MYC rearrangements that do not involve the Ig heavy or light chain locus (13). The clinical consequences of secondary MYC aberrations are not known, but it is known that they are associated with more proliferative disease. Chromosome 13 aberrations Chromosome 13 aberrations are found in about 50% of cases, with most being complete monosomy 13 (85%), while the remaining 15% constitute deletion 13. The first link between a recurrent chromosome abnormality and prognosis in MM was identified when monosomy and/or del 13 were associated with aggressive clinical course (45e51). Historically, del(13) has been associated with an unfavorable prognosis in MM, but there is now increasing evidence that its prognostic relevance may be related to its association with other genetic aberrations. Recent studies suggest that the prognostic significance of monosomy 13 may emanate from its close association with the t(4;14)(p16;q32) (34,38). Fonseca et al. (34) suggest a crucial role for chromosome 13 deletions as a prerequisite for the clonal expansion of myeloma tumors. Deletions of 17p13 The deletion of 17p13 in MM presumably leads to the loss of heterozygosity of TP53, a well-characterized tumor suppressor gene that transcriptionally regulates cell-cycle progression and apoptosis. The deletion or inactivation of TP53 is a rare late event, with deletions of 17p13 being reported in 10% of patients in interphase FISH studies (52e55). The deletion 17p13 has been identified clinically as an indicator of very poor prognosis (34,39,41,55). In fact, the 17p deletion is sited as the most important molecular cytogenetic factor for prognostication yet identified in interphase FISH (34). It has been reported that patients with 17p deletions have more aggressive disease, a higher prevalence of extramedullary disease, and overall shorter survival (39,41,55). FISH studies of clonal evolution in myeloma indicate that the deletion occurs most commonly in subclones (56). Interestingly, it has also been reported that in rare cases, the loss of 17p13 can occur as a result of unbalanced translocations of 1q12 to 17p (40,41). This type of aberration results in the simultaneous gain of 1q and deletion of 17p13. A model for the clonal evolution of MM based on interphase FISH studies, proposed by Cremer and colleagues (56), provides evidence for the timing of the cytogenetic aberrations. They used a comprehensive probe set of 10 chromosome-specific probes and two IgH rearrangements to identify associations of chromosome aberrations in newly diagnosed patients. In this model, clustering analysis indicated four major branches of an oncogenetic tree, including a hyperdiploid branch, a del(13) and t(4;14), a t(11;14) branch, and a gain of 1q branch. Statistical modeling of their

6 J.R. Sawyer oncogenetic tree indicated that early independent events were gains of 15, 9, 11 and t(11;14), deletion of 13q followed by t(4;14), and finally, gain of 1q. In this model, aberrations of 17p, 22q, 8p, and 6q were subsequent events. The t(4;14) translocation was associated with the non-hyperdiploid pathway and was always preceded by deletion 13q. The deletions of 6q, 8p, and 22q were linked to the major branch of non-hyperdiploid MM (56). This model also indicates deletion of 17p13 as a late secondary event in MM. In the pathway they describe leading to hyperdiploidy, gains of 9 and 15 were associated, while gain of 19 was a subsequent event. Interestingly, they found that gain of 1q and deletion of 17p occur at approximately the same frequency in both the hyperdiploid and nonhyperdiploid groups. This interphase FISH study supports the framework provided by conventional cytogenetic studies and provides evidence of distinct subgroups of MM. Chromosome 1 aberrations Chromosome 1 aberrations are the most common structural aberrations in MM and mostly involve deletions in 1p and amplifications in 1q (57e64). The deletions of 1p are mainly interstitial deletions that have been characterized by banding and FISH studies (60e64). The 1p deletions are defined by varying interstitial deletions of the region spanning 1p13w1p31. Deletions of 1p are associated with a poor prognosis (60,63). Also, in the short arm of chromosome 1, a rare recurring reciprocal translocation has been reported involving MYC (13,19). The t(1;8)(p12;q24) juxtaposes MYC to 1p12 and apparently occurs as a secondary aberration. This aberration represents an example of a MYC translocation that does not involve an immunoglobulin rearrangement. The clinical significance of this translocation is not known. It has been noted that during karyotypic evolution, the der(1)t(1;8)(p13;q24) chromosome can become duplicated, resulting in extra copies of both MYC and 1q21 (Sawyer JR, unpublished data). Chromosome 1 aberrations involving the q arm are particularly complex and tend to become unstable during tumor progression.the 1q aberrations were first reported by conventional cytogenetics, and include direct and inverted duplications of 1q12wq23, isochromosomes 1q, and both whole-arm and/or jumping translocations of 1q (4e9,57,58). The amplification of the proximal 1q21 region in MM has been reported by FISH in about 40% of patients with newly diagnosed MM, and in 70% of patients at relapse (64). The 1q arm, particularly the 1q12wq23 region, contains a large number of possible candidate genes that show amplification and/or deregulated expression important in myeloma, including MUC1, MCL1, PDZK1, IL6R, BCL9, CKS1B, PSMD4, UBAP2L, UBE2Q1, and ANP32E, among others (65e76). The mechanisms for the amplification of 1q are believed to involve 1q12 pericentromeric instability, which most commonly increases the copy number of 1q by direct (Figure 1A) and/or inverted duplications (Figure 1B) (57,58,77e82). It is noteworthy that further instability of the 1q12 pericentromeric heterochromatin can result in adding whole-arm segments of 1q to non-homologous chromosomes by jumping translocations of 1q (79). In the jumping 1q rearrangements, the whole arm of 1q is the donor chromosome, and any other chromosome can become a potential receptor chromosome (81). Two types of jumping 1qs are common. The first type is the telomeric jumping 1q, in which the 1q translocates to the telomere of a receptor chromosome. The second type occurs when the 1q translocates to the pericentromeric region of a receptor chromosome. This second type results in whole-arm deletions in the receptor chromosome. Two recurring whole-arm unbalanced pericentromeric translocations of 1q translocations are most commonly reported in MM. These include the der(1;16)(q10;p10), which results in the gain of 1q and loss of all of 16q (Figure 1C), and the der(1;19)(q10;p10) (Figure 1D), which results in the gain of 1q and loss of 19q (7,58). Multiple receptor chromosomes can be involved in both whole-arm and telomeric 1q translocations within a single clone, demonstrating the higher level of amplification and instability introduced by the jumping 1q aberrations (Figure 1E). Interestingly, a number of deletions reported in myeloma, such as deletions of 6q, 8p, and 17p, have all been reported to be associated with unbalanced whole-arm jumping 1q translocations (57,58). The clinical significance of these unbalanced whole-arm aberrations has not been established, but these types of 1q rearrangements are apparently favored during tumor progression, providing a selective advantage to subclones containing them. A mechanism for the amplification of a 1q12wq23 amplicon has been described recently in MM (80). The mechanism involves breakageefusionebridge (BFB) cycles of the 1q12 pericentromeric heterochromatin and the adjacent bands of 1q, resulting in an inverted repeat pattern of amplification of the 1q12wq23 region (Figure 1F). The BFB cycles amplify a large number of genes within an amplicon of about 10e15 megabases spanning the 1q12wq23 region (71,80). The bracketing of the amplicon by 1q12 pericentromeric heterochromatin apparently facilitates the subsequent translocation of segments of this amplified region to other chromosomes similar to the jumping whole arm 1q. Copies of the 1q12wq23 amplicon can become integrated in complex multi-chromosome translocations during tumor progression. Amplification of 1q is universally identified in genetic studies of MM and is associated with poor prognosis according to most studies. However, molecular profiles have identified a number of different genes believed to be of importance in this region (65e74). Because of the lack of a highly focal lesion and the lack of consensus on any critical genes, the clinical significance of amplification 1q is controversial. The differences in genes identified and clinical outcome may be related to differences in sample populations and data-mining approaches (8) or, alternatively, perhaps the 1q amplification is simply a surrogate marker of more clonally advanced and genomically unstable tumors (83). More research is needed to clarify the meaning of amplification of 1q, but the 1q aberrations seem to introduce an increased level of genetic instability in MM, which follows the concept that only chromosome aberrations that confer a growth advantage to malignant cells would spread within the tumor clone (84). MDS type chromosome aberrations in MM Myelodysplastic syndrome (MDS) is a well-recognized complication of cancer chemotherapy. Alkylating agents have traditionally been used in the management of MM as

Cytogenetic and molecular classifications in multiple myeloma 7 Figure 1 Representative partial karyotypes of metaphase chromosomes demonstrating the different types and degrees of amplification of chromosome 1. FISH probes for 1q12 (red), 1q21 (green), and 16q11 (aqua) are shown on inverted DAPI images of chromosomes depicting G-band patterns. (A) Chromosome 1 pair showing interstitial deletion of 1p (arrow) in the homologue on left and a direct dup1q12wq23 on chromosome 1 on the right. Note two copies of probes satii/iii and 1q21 (arrows) on the duplicated 1. (B) Chromosomes 1 showing the normal homologue on the left and the abnormal homologue on the right, demonstrating both an interstitial deletion of 1p (arrow) and amplification of 1q in the same chromosome. Note four copies of 1q21 (arrows) in an inverted duplication pattern. (C) Example of an unbalanced whole-arm translocation of 1q to chromosome 16q. Chromosomes 1 are on the left, and chromosomes 16 on the right. Aqua probe denotes 16q11 heterochromatin. Note the loss of 16q distal to the aqua probe on the der(1;16)(q10;p10). The entire long arm 1q is translocated to the pericentromeric region of 16q, and a total of three copies of 1q21 (arrows) are present. (D) Example of an unbalanced whole-arm translocation of 1q to 19q. Note that the result of this translocation is the der(1;19)(q10;p10) chromosome, which shows an extra copy of 1q21 (arrows) and loss of the entire 19q. (E) Example of jumping 1q, in which all or part of 1q is translocated to three different receptors, including chromosomes 19, 21, and 22. Note the inverted duplication (dup) on chromosome 1 (second from left) and copies of 1q (arrows) on the three different nonhomologous chromosomes. The whole-arm der(19)(q10;p10) in this case is the same type seen in the patient in (D). The der(21) results from a segmental translocation of the inverted dup of 1q to the short arm of the 21. The der(22) results from a whole-arm 1q translocated to short arm of 22. (F) Homologues of chromosome 1 demonstrating amplification of 1q12wq23 by breakage-fusion-bridge (BFB) cycles. Note multiple copies of 1q21 (arrows) on the abnormal homologue on the right. The copies of the 1q12wq23 amplicon occur in an inverted repeat pattern, with a deletion of the 1q distal to the amplified region. Dotted lines between normal homologue 1 (left) and abnormal homologue denote the size of expansion of the 1q12wq23 region by BFB cycles. well as other malignant diseases. Cytogenetic aberrations typifying MDS after alkylator-based therapy include partial or complete deletions of 5, 7, and 20, as well as þ8, and whole-arm t(1;7)(q10;p10). Other chemotherapy drugs such as topoisomerase inhibitors, estoposide, and doxorubicin target chromosome 11 (85). In MM, MDS-type chromosome

8 J.R. Sawyer aberrations are found after high-dose therapy, and they demonstrate distinct differences from the complexity and ploidy levels normally found. This feature can be used to distinguish between MM and MDS cytogenetic aberrations (86). MDS-type chromosome aberrations occur in MM both as sole aberrations in individual MDS clones and also within the typical otherwise complex myeloma karyotypes (87,88). After high-dose therapy, karyotypes with þ8 and deletions of 5q, 7q, and 20q are the MDS aberrations observed most frequently (88). Nilsson et al. (86) reported 20qe in separate clones occurring in 10% of karyotypically abnormal MM/ MGUS patients. In a large prospective study for the detection of MDS abnormalities, Barlogie and colleagues (89) noted aberrations in 4% of patients after high-dose therapy. Of the patients showings MDS aberrations, patients most commonly developed del(20q) (25%), e7/7qe (16%), t(1;7) (q10;p10) (13%), e5/5qe (10%), del(13q) (7%), þ8 (5%), and del(11q) (2%) (89). Molecular classifications of multiple myeloma Whole genome molecular profiling techniques have ushered in a new era in the detection of genomic defects (90). These techniques use different approaches, but all provide dramatically increased resolution to the analysis of the genomic aberrations (91e102). Gene expression profiling (GEP) has enabled the simultaneous analysis of RNA expression patterns of thousands of different genes pertinent to biologic functions. GEP has been shown to be a tool that can reproducibly demonstrate molecular subclassifications of MM. DNA copy number (CN) abnormalities detectable by both highresolution array comparative genomic hybridization (acgh) and single-nucleotide polymorphism (SNP)-based arrays have also demonstrated evidence of their importance in the molecular classification of MM. The SNP-based analysis has an added advantage in that it allows not only the identification of CN changes, but also the identification of loss of heterozygosity (LOH) due to monosomy and uniparental disomy (UPD). UPD is not detectable by conventional cytogenetics, FISH, or acgh. The first molecular classification system to be described in MM was the Translocation and Cyclin D (TC) classification system proposed by Bergsagel and colleagues (94). This system is based on GEP of mrna spikes involving five recurrent IGH translocations, specific trisomies, and the overexpression of cyclin D (CCND) genes. The system proposes eight TC groups with significant uniform differences in global gene expression profiles and clinical features (94). The TC groups include those with primary translocations of 4p16, 11q13, 6p21, and 16q23, and those that over-express CCND1 and CCND2, either alone or in combination. Rare cases in this system do not over-express any CCND genes (80). The TC classification focuses on the different mechanisms that dysregulate CCND genes, and suggests that cyclin D dysregulation is an early and unifying pathogenic event in myeloma. Patients in the TC 4p16 group have substantially shortened survival, as do patients in the TC 16q23 (MAF ) group. In contrast, patients in the TC 11q13 group appear to have a better survival. A second GEP-based classification system developed by Zhan et al. (96) divides MM into seven different classes based on the presence of translocations or hyperdiploidy. The classes include an MS class characterized by the overexpression of MMSET and FGFR3 involving the t(4;14), and an MF class that identifies the over-expression of both MAF/MAFB, resulting from the t(14;16) and t(14;20). CD-1 and CD-2 classes in this system identify overexpression of CCND1 and CCND3 genes and the t(11;14)(q13;q32) and t(6;14)(p21;q32), respectively. The HY class represents the hyperdiploid myeloma with the odd trisomies of 3, 5, 7, 9, 11, 15, 19, and 21. The LB class is characterized by lower expression of genes involved in bone disease, and low incidence of MRI-defined focal bone lesions. Finally, the PR class is characterized by the over-expression of cell cycle progression and cell proliferation genes (96). The PR class contains a high percentage of cases with metaphase chromosome aberrations and is associated with poorer survival than the other classes. In this system, the MS class is also a high-risk entity with poor prognosis. Using this model, Shaughnessy and colleagues (73) provided the first validated classifier for prognosis predication in patients with uniformly treated myeloma. This group identified a subset of 70 genes whose up-regulation is sufficient to predict poor prognosis. Thirty percent of the 70 genes identified as important are located on chromosome 1, with most of the down-regulated genes on 1p and most of the up-regulated genes on 1q. This group further simplified the subset of 70 genes to a list of 17 genes capable of providing the same prognostic discrimination. This system identifies the main genetic subtypes of MM and identifies the PR, MS, and MF groups as patients with poor prognosis, making this GEP-base system clinically relevant (73). More recently, Decaux and colleagues (76) have also proposed a GEP-based model that is predicative of outcome. Using a top-down supervised approach in newly diagnosed patients, they developed a 15-gene model that revealed cell cycle and chromosomal instability signatures. They identified two groups: a high-risk group involving a homogeneous biologic entity, and a more heterogeneous low-risk group. Their analysis found that myeloma cells in high-risk patients over-expressed genes involved in mitosis and its surveillance, whereas low-risk patients included the hyperdiploid gene signature. The novelty of their findings is the specific enrichment of mitotic-phase genes in patients with aggressive disease. Both this 15-gene model and the 17-gene models (73) have identified cell cycle genes as important in proliferative tumors, and both are powerful and both have prognostic significance in high-risk patients. In a study using high density SNP arrays, Avet-Loiseau et al. (102) identified recurrent copy number aberrations associated with prognosis. The copy number changes involving amplifications in 1q and deletions in 1p, 12p, 14q, 16q, and 22q were most frequently associated with adverse prognosis. Conversely, amplifications of chromosome 5, 9, 11, 15, and 19 conferred a favorable prognosis. This study identified patients with del(12p) alone, or amp (5q) and del (12p) as being associated with very poor outcome. Integration of profiling techniques The integration of data from the different molecular approaches, including interphase FISH, GEP, acgh, and

Cytogenetic and molecular classifications in multiple myeloma 9 Figure 2 Summary of genomic profiles and recurrence of chromosomal alterations in primary tumors demonstrated by acgh. The recurrence plot mirrors the frequencies of previously reported chromosomal gains and losses, including the deletions of 1p and amplifications of 1q. Integer-value recurrence of copy number aberrations across the samples in segmented data is plotted on the y axis. The x axis is in chromosomal order. Dark red or green bands denote the number of samples with gain or loss of chromosome material, and bright red or green bars represent the number of samples showing amplification or deletion. Asterisks show focal deletions of the kappa (2p;12), IgH (14q32), and lambda chain (22q11) loci physiological in B cell postgerminal center neoplasms [used with permission, Carrasco et al. (71)]. SNP arrays have been used to identify subgroups within previously identified ploidy classifications. Integrating acgh and GEP studies, Carrasco et al. (71) identified genomically distinct subtypes within hyperdiploid MM, revealing a previously unrecognized level of molecular heterogeneity within the karyotypes. A recurrence plot of their acgh data (Figure 2) mirrors the frequencies of previously reported chromosomal gains, including 1q, 3, 5, 7, 9, 11, 15, 19, and 21. Also identified are losses, including interstitial deletions of 1p, loss of 13, and deletions of 16q. In this study, one hyperdiploid group with the odd number trisomies and chromosome 11 gains conferred a more favorable outcome, while the other hyperdiploid group that lacked chromosome 7 and 11 trisomies and showed the presence of 1q gain and e13 had a poor prognosis. In addition, this study also provided molecular evidence for the importance of genes in the 1q12wq23 region (71). This group defined a region of proximal 1q with a marked enrichment of genes showing a gain and/or amplification that spans a region of 10e15 Mb corresponding to a 1q21wq23 amplicon in MM. Walker et al. (72) used global SNP mapping and GEP, and for the first time identified UPD affecting a number of chromosomes. UPD is identified by allelic loss that occurs when one allele is deleted and the remaining dysfunctional allele is duplicated, resulting in loss of heterozygosity (LOH) without loss of copy number. The most frequent alterations involving LOH included 1p, 1q, 6q, 8p, 13q, and 16q. They provide evidence that UPD is prevalent in MM and occurs through multiple mechanisms, including mitotic nondisjunction and mitotic recombination. It is of interest that they found uniparental trisomy of 1q occurring by mitotic recombination. Finally, Walker and colleagues (63) have recently compiled a compendium of SNP copy number aberrations and their prognostic value. They identified copy number neutral LOH or UPD on 1q (8%), 16q (9%), and X (20%). In addition, they identified deletions of 4p occurring in about 25% of the t(4;14) samples, which results in the loss of the over-expressed FGFR3 allele. It is important to point out that this study also re-examined the prognostic implications of 1q amplification by removing cases with the most adverse cytogenetic factors. These aberrations included translocations involving FGFR3/ MMSET, MAF, and MAFB, and del17p, after which the amp 1q still retained prognostic significance. This study also confirmed the presence of a minimally amplified region on 1q between 1q21 and q23 (71). Based on the combined use of SNP and expression quartile analysis, they identified genes with adverse prognostic significance on chromosomes 1p, 1q, and 17p. Summary and further studies Genetic subtypes of MM have been identified which have different underlying biologic features and show heterogeneity in clinical outcomes. 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