Genetics of multiple myeloma

Best Practice & Research Clinical Haematology Vol. 18, No. 4, pp. 525–536, 2005 doi:10.1016/j.beha.2005.01.006 available online at http://www.sciencedirect.com

3 Genetics of multiple myeloma Michaela J. Higgins Rafael Fonseca* MD Department of Internal Medicine, Division of Hematology and Oncology, Comprehensive Cancer Center, Mayo Clinic, 13400 E Shea Blvd, Scottsdale, AZ 85258, USA In recent years, we have seen an explosion in knowledge of the genetics and cytogenetics of the plasma-cell neoplasms. This chapter will deal with these advances and will place them in the integrative context of the pathophysiologic basis of the disease, and will discuss the important clinical implications of these abnormalities. We have learned that myeloma can be classified into increasingly definable subgroups that follow a basic global hierarchical grouping. All gene expression profiling strategies have come to similar conclusions and confirm the subgroups previously identified by karyotype, molecular cytogenetics and other genetic studies. At the top level there are two major pathogenetic pathways for the development of plasma cell tumors: one that is associated with hyperdiploidy and one that is characterized by the presence of chromosome translocations involving the immunoglobulin heavy chain locus (IgH). These translocations are seen in up to 60% of patients, but involve a common recurrent chromosome partner in only 40–50% of patients. Several genetic markers are now shown to be associated with a shortened survival. Of these, the most common ones include abnormalities (deletion and monosomy) of chromosome 13, the global state of hypodiploidy and abnormalities of chromosome 1. Many of the translocations observed in MM are also seen in monoclonal gammopathy of undetermined significance (MGUS), even in individuals without progression to full malignant disease for many years. The identification of critical genetic lesions will pave the way for the development of disease-targeted therapy. Key words: myeloma; genetics; cytogenetics; pathogenesis; prognosis.

Recent investigations have shown that all myeloma clonal plasma cells harbor an array of complex cytogenetic and genetic abnormalities. The abnormalities present in these cells have been identified as the founder genetic lesions for the disease. These aberrations, initially thought to occur in a chaotic and random fashion, seem to follow previously unsuspected relationships. The specific chromosomal abnormalities in the plasma-cell neoplasms impart important features to the cells, allowing the molecular * Corresponding author. Tel.: C1 480 301 6118; Fax: C1 480 301 9162. E-mail address: [email protected] (R. Fonseca). 1521-6926/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved.

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cytogenetic classification to divide the disease into discrete subgroups. These chromosome aberrations have gained widespread acceptance as biologic markers and are gaining increasing acceptance as factors for clinical decision-making in the disease.

BACKGROUND AND TECHNICAL ASPECTS Many of the novel techniques for the analysis of cancerous cells do not depend on the mitotic proliferation for analysis. Accordingly techniques designed for the analysis of the transcriptome (RNA gene expression profiling) or numerical and structural chromosome abnormalities (array-based comparative genomic hybridization and interphase FISH, respectively) have largely replaced older techniques of genetic assessment. The technical complexities associated with the performance of these tests, however, have limited their widespread applicability in routine clinical practice. Many of these techniques involve the selection of cells for DNA or RNA extraction, or coperformance of testing with immunofluorescent detection methods. It is critical to emphasize that for the correct analysis of cells in myeloma one must restrict the analysis to the myeloma cells. As mentioned before, this can be done by cell purification or by co-labeling by immunofluorescence of the cells. In the absence of these steps the genomic results at large are generally unreliable. Because of this lag in clinical applications many centers still perform conventional karyotype analysis for the prognostic evaluation and clinical classification of patients. The information obtained by this methodology is very important but limited because of the low proliferative potential of plasma cells and thus the low percentage of cases with abnormalities noted (informative karyotypes seen in only 15–30% of patients).1 In the vast majority of cases, when one orders a karyotype analysis the results come back as normal, with metaphases originating from myeloid elements of the bone marrow.1–9 Another limitation is that some abnormalities—such as the t(4;14)(p16.3;q32)-are cryptic (see below).

SPECIFIC CHROMOSOME ABNORMALITIES Aneuploidy Specific chromosome changes Four major subtypes of ploidy categories exist in multiple myeloma (MM): hypodiploid, pseudodiploid, hyperdiploid and hypotetraploid or near tetraploid.6–8,10 The hyperdiploid cases are the only ones that stand out from the rest, mainly because they are categorized by multiple trisomies and have a chromosome count close to 53 chromosomes. These trisomies involve chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, but notably lack trisomies of chromosome 13.1–3,5,8,11–15 Monosomies are seen in many cases of MM, but are most prominent in the case of hypodiploid MM. The most common monosomies are those of chromosomes 13 and 14 (highly associated together in hypodiploid variant MM).4,11,16–21

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Hyperdiploid versus non-hyperdiploid MM The aforementioned four major subtypes of ploidy categories existing in MM— hypodiploid, pseudodiploid, hyperdiploid and hypotetraploid (also called neartetraploid)6–8,10—have been recently condensed into two major categories: the hyperdiploid myeloma and the non-hyperdiploid myeloma.6–8 The biologic basis of this is that the non-hyperdiploid MM group harbors a large percentage of patients with primary IgH translocations (O85%), while these translocations are far less common in patients with hyperdiploidy (!30% of patients).8,22,23 Chromosome 13 abnormalities are also more common in patients with non-hyperdiploid myeloma. The ploidy categories of myeloma can be detected by karyotype analysis (low sensitivity), cIg-FISH (technically demanding), gene expression profiling (not widely available) or PI DNA index (by flow cytometry).24,25 While this last method is capable of determining hyperdiploidy patients from all others it is not capable of discriminating patients with hypodiploidy from those with pseudodiploid karyotypes.16,26–28 Clinical implications of ploidy categories While trisomies have been associated with an improved outcome in MM, this is probably because of their association with the hyperdiploid variant myeloma, which in some studies has been associated with an improved survival.29 The only other important clinical association with ploidy category is that of an adverse outcome for patients with hypodiploidy.7,8,26,30–32 Hypodiploidy is a powerful prognostic marker that has independent prognostic implications.7,8,32 The specific contribution and pathogenetic significance of hypodiploidy has not been elucidated.22,23 In particular it appears that some of the high-risk translocations are associated with this hypodiploidy state. Chromosome 13 abnormalities (D13) Biology of chromosome 13 abnormalities Chromosome 13 abnormalities (jointly referred to as D13) are observed in one half of patients with myeloma33–39 and in 50% of abnormal karyotypes.5,40,41 With the use of interphase FISH, it is now clear that D13 occur in all stages of the PC neoplasms, including monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma SMM.33,36–38 While conflicting data exist regarding the actual proportion of cells with these abnormalities, they seem to be clonally selected and present in the majority of the clonal cells.33,36,42,43 The actual percentage of cases with MGUS and D13 is still being investigated, but its prevalence is likely close to that reported in myeloma.33,36,43 Initial studies suggested that chromosome 13 abnormalities would be involved as a progression event from MGUS to myeloma44; more recent studies suggest that this is likely not the case.45 In serial cases of MGUS evolving to myeloma, D13 abnormality was present since the MGUS state.45 In most cases D13 represent monosomies, and in the remaining 15% of cases they are interstitial deletions, mostly localized in band 13q14.2,5,40,46–48 Is it causative? Chromosome 13 karyotypic abnormalities have been observed in a large proportion of human cancers. With the exception of point mutations of the Rb gene in hereditary and sporadic retinoblastoma and in cases of osteosarcoma, no specific abnormalities are

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associated with oncogenesis. However, in many cases—such as myeloma and B-cell chronic lymphocytic leukemia-chromosome 13 still appears to have important clinical associations. In the case of myeloma, many pieces of indirect evidence further support a specific biologic role for the abnormality.39 These include the characteristic effects observed in gene expression profiling, its reported recurrent prevalence, its clonal selection, its clinical implications, its association with other primary cytogenetic lesions and the rarity of chromosome 13 trisomies.2,14,37–40,46–51 Clinical and pathologic implications including prognosis A key observation that rekindled interest in the field of myeloma genetics was the report of negative prognostic implications of D13 detected by karyotype.49,52,53 In all studies to date D13 are associated with a shorter survival and lower likelihood of response to treatment.29,37–39,49,50,54 These reports include patients treated with either conventional chemotherapeutic agents or with high-dose therapy. The efficacy of the novel agents now available for the treatment of MM in patients with chromosome 13 abnormalities is as yet unknown. While the net effect of D13 is greater when D13 is detected by karyotype (i.e. higher hazards ratio) it is also an important and independent prognostic factor when it is detected by interphase FISH.29,37–39,49,50,54 D13 in combination with the b2-microglobulin is currently being used by the Intergroup Francophone du Myelome (IFM) to stratify patients entered into clinical trials designed for patients with aggressive disease. Integration of D13 into mainstream clinical practice has occurred over the last 3 years, but many questions remain. For instance, the retrospective comparison between the data published by the Eastern Cooperative Oncology Group and the IFM suggest that the net benefit of high-dose chemotherapy is greater for patients who do not have D13 (detected by FISH)38,39, suggesting limited clinical benefit of high-dose therapy for patients with D13. One study found that the use of interferon-a in patients with D13 resulted in a shorter survival.39 Immunoglobulin heavy chain (IgH) locus translocations Biology of IgH translocations IgH translocations are believed to be seminal events in the pathogenesis of one half of plasma-cell neoplasms (Table 1).55,56 The IgH translocations in myeloma always result in

Table 1. Prevalence and clinical importance of IgH translocations. Abnormality IgL translocations IgH translocations t(4;14)(p16.3;q32) t(11;14)(q13;q32) t(14;16)(q32;q23) Cyclin D3 other or unknown e.g. t(6;14)(p21;q32) Other IgH

MGUS and SMM (%)

MM (%)

Upregulated oncogenes

Effect on prognosis

!20 35–50 2–10 15–30 2–5 ?

!20 50–70 15 16 5 4

c-myc and others See below FGFR3 and MMSET Cyclin D1 and myeov C-maf WWOX? Cyclin D3 other

Unknown Mixed Adverse Favorable Adverse Unknown

10–15

15–30

mafB MUM1 Others

Unknown

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the increased transcription of oncogenes (mostly proliferation genes and transcription factors) and do not result in fusion proteins.57 They are present in 50–60% of patients, with a slight increase in prevalence with the more advanced stages of the disease. They are particularly common in patients with plasma-cell leukemia and human myeloma cell lines, likely indicating a selection bias among those patients for cells that can survive outside of the bone-marrow microenvironment.58–60 Limited studies indicate that translocations involving other immunoglobulin loci are also present, but only involve the l light-chain locus (IgL-l seen in 10% of patients) and almost never in the k locus.36 For the most part IgH translocations are mutually exclusive (no more than one translocation present in one patient sample) but they are almost always clonally selected (present in close to 100% of the clonal cells). In sharp contrast with other B-cell neoplasms, IgH translocations in myeloma involve an array of chromosomal partners.57 Three main types of IgH chromosomal translocations are observed in myeloma: (1) those involving the cyclin D genes: 11q13 (cyclin D1) in 15% and 6p21 (cyclin D3) in another 5% of cases, translocations involving cyclin D2 just being described unpublished observations; (2) translocations involving the up-regulation of FGFR3 and MMSET (seen in w15% of cases); and (3) translocations involving MAF genes: t(14;16)(q32;q23) with c-maf up-regulation and 20q11 (maf-B) in another 3% of cases. These three main groups compromise 45% of all myeloma cases and have been referred to as the ‘primary IgH translocations’. In addition, another 20% of MM cases have translocations that involve other chromosome partners that occur at a prevalence of 1% or less.58,59 These have been referred to as the ‘secondary IgH translocations’. It is possible that the IgH locus, even after sustaining a translocation, remains fundamentally unstable and prone to undergoing additional chromosomal translocations. Most translocations in myeloma are thought to occur during the process of class switching, with a few occurring at other times of B-cell maturation.57 Translocation breakpoints involving the partner chromosomes are highly variable, with large genomic regions involved. Lastly it should be noted that IgH translocations are highly conserved in the process of disease evolution. It is now believed that translocations present at any time point in the course of a particular patient’s disease will be present at subsequent (and previous) time points. IgH translocations in MGUS IgH translocations are early genetic lesions and the prevalence in MGUS is nearly as high as in MM. Because of technical limitations it has been hard to elucidate the actual proportion of clonal cells in MGUS that harbor IgH translocations.33,36 While details of the specific prevalence of the individual chromosome partners are still being elucidated, we know that all IgH translocations reported in myeloma can also be observed in MGUS.36,59,61,62 Microenvironment dependence Several observations suggest that myeloma with IgH translocations may be better able to survive outside of the bone-marrow microenvironment. We have previously noted that IgH translocations are highly prevalent in the human MM cell lines (HMCLs), and in the case of plasma-cell leukaemia.56,63,64 Virtually all HMCLs are generated from primary PCLs or extramedullary disease. It is precisely these patient groups that harbor a high prevalence of IgH translocations. Cell lines derived from patients with no IgH translocations seem to be quite rare and have yet to be convincingly described.

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These cell lines form an excellent model for the ex-vivo study of the disease, but are not representative of patients without the IgH translocations. p53 deletions In most patients, there are no deletions or mutations of p53. When tested by FISH, deletions of the p53 locus, 17p13, detected by FISH are reported in 10% of patients by most studies.59,65–67 The presence of the gene deletion is an independent predictor of shortened progression-free or overall survival after either conventional-dose chemotherapy or high-dose chemotherapy and autologous stem-cell transplantation.1,2 In addition, patients with this specific abnormality are more likely to have other features of aggressiveness such as hypercalcemia, high serum creatinine levels and extramedullary plasmacytomas.2,3 The prognostic implications of p53 mutations are currently unknown. Chromosome 1 abnormalities Chromosome 1 abnormalities are very common in most hematologic malignancies and constitute the most common structural aberration in MM. They have been observed in up to 48% of patients.5,68 Until recently no specific gene has been linked with chromosome 1 abnormalities, which is surprising given the wide range of breakpoints found on both the p and q arms. Inoue et al recently reported that amplifications of 1q may be associated with more aggressive phenotypes.69 Monosomy of chromosome 1 is seldom observed. We have seen that structural abnormalities of chromosome 1 are associated with elevated plasma-cell labeling index and significantly shortened survival in myeloma.8 Inoue and colleagues have similarly found that patients with 1q amplifications that track with 1p deletions are also associated with more aggressive disease.69

CYTOGENETIC SUBGROUPS OF PATIENTS While multiple classification schemes have been proposed for the molecular genetic and cytogenetic classification of myeloma, most are focused on the underlying premise of the importance of the baseline cytogenetic features of patients. A recently proposed classification is based solely on the presence of cytogenetic aberrations detected by FISH.59 This classification is able to identify groups of patients with dissimilar clinical, phenotypic and outcome features. A molecular refinement of this classification is also proposed, and considers only the translocation status of a patient and the level of expression of cyclin D genes (TC classification).57 These classification schemes have led to the identification of unique groups of patients with similarities defined by the chromosome abnormalities. The groups are composed as follows. Plasma cell tumors with t(11;14)(q13;q32) The t(11;14)(q13;q32) results in up-regulation of cyclin D170 and is observed in 15% of myeloma cases.58,71 This translocation is also seen in patients with MGUS (15–30%) and AL amyloidosis (!50%).33,36,72–74 Because of the lower prevalence of this abnormality in the early stages of the plasma-cell neoplasms, we have suggested that it may be

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negatively selected for progression to myeloma.36,72–74 The t(11;14)(q13;q32) is found in a high proportion of patients with light-chain amyloidosis and IgM variant MM.72,73,75 The over-representation of the t(11;14)(q13;q32) in human MM cell lines makes it likely that it is prone to result in aggressive clonal growth if the needed secondary genetic aberrations are also present or acquired.70 While cyclin D1 may be up-regulated at low levels by trisomy of chromosome 11, the level of cyclin D1 expression is much higher in patients with t(11;14)(q13;q32). We have reported on the association between the t(11;14)(q13;q32) and lymphoplasmacytic morphology76, and others have reported a small mature plasmacell morphology.77 The t(11;14)(q13;q32) is clearly associated with cell-surface expression of CD20. In fact the majority of CD20 expressing myeloma (15% of cases) greatly overlaps with patients having this translocation (also 15% of cases).77 In one study, 10 of 12 patients with CD20C MM had t(11;14) (83%) as opposed to five of 54 (9%) CD20C patients (P!0.001). Patients with the t(11;14)(q13;q32) are mostly non-hyperdiploid, with variations from the 2N and 4N karyotypes being extremely rare. It is now well established that the t(11;14)(q13;q32) is not associated with shortened survival, and if anything an improvement in survival is suggested. This improvement appears to be more pronounced in some series among patients treated with high-dose therapy.61,71,78 We have been unable to confirm this observation, suggesting that the improvement may be at best modest (unpublished information, Rafael Fonseca, December 2004). Plasma cell neoplasms with t(4;14)(p16.3;q32) The t(4;14)(p16.3;q32) was first identified in human myeloma cell lines, and is present in 15% of myeloma samples.61,74,79–82 The two target genes up-regulated by the t(4;14)(p16.3;q32) are FGFR3 and MMSET. The t(4;14)(p16.3;q32) has been observed by some investigators in cases of MGUS and smoldering myeloma, thus indicating its insufficiency for conferring full malignant potential to the clone.36,58,62,74,83,84 Likewise the abnormality has been found in cases of light-chain-associated amyloidosis at a proportion similar to that reported in myeloma.84 Almost all patients with t(4;14)(p16.3;q32) also harbor D13.58,79 This tight correlation has made the dissection of the contribution of the t(4;14)(p16.3;q32) versus that of D13 difficult to interpret. In 25% of cases with the t(4;14)(p16.3;q32) the derivative chromosome 14 is lost (harboring the translocated FGFR3 allele). The opposite, loss of the derivative 4 (harboring the MMSET translocated allele), has not yet been reported.59,62,85 These observations suggest a greater role for MMSET in the perpetuation of the clonal expansion of cells59,62,85, but it is certainly possible that deregulation of both FGFR3 and MMSET are important initiating events. It is now widely accepted that t(4;14)(p16.3;q32) is an unfavorable prognostic factor in myeloma independent of the mode of treatment59,61,62, although some have suggested this effect is independent of the expression of FGFR3.62 There may also be a tight correlation between hypodiploidy and t(4;14)(p16.3;q32). Plasma cell tumors with hyperdiploidy Hyperdiploidy is found in a large heterogeneous group of patients comprising 40–60% of all myeloma patients.2,5 The primary genetic lesions causing hyperdiploidy are unknown.

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Hyperdiploidy can be referred to as a category of the disease (patients with multiple trisomies and no IgH translocations) or as a feature of a given tumor (e.g. up to 30% of patients with t(4;14)(p16.3;q32) have hyperdiploidy). The prevalence of hyperdiploidy is independent of stage and is also observed in MGUS. Hyperdiploidy has been associated with an improved outcome probably related to the low prevalence of IgH translocations.

A UNIFYING THEORY? Most of the chromosome translocations in myeloma result in up-regulation of proliferation genes. It has been observed that in almost all cases there is up-regulation of at least one of the three cyclin D genes, suggesting that early dysregulation of these genes may represent the unifying oncogenetic event. This classification proposes that: (1) cyclin D1 can be up-regulated by either a chromosomal translocation (high level; 11q13) or by a trisomy (low level; D1); (2) in some patients there is cyclin D2 upregulation as a consequence of the t(4;14)(p16.3;q32) or the t(14;16)(q32;q23) or as a primary event (D2) or in conjunction with low cyclin D1 expressing tumors (D1CD2); and lastly (3) cyclin D3 is up-regulated by direct translocations involving its loci (D3). Many more questions remain, in particular those that are needed for the full understanding of hyperdiploid variant myeloma.

SUMMARY The study of the genetics and cytogenetics of myeloma has seen remarkable progress in the last 10 years. These studies have modes from the discovery base to the current situation where genetic knowledge is integrated into clinical practice. It is now recognized that myeloma is composed of two main types of disease: one that has hyperdiploidy and another that harbors a higher incidence of IgH translocations. Each one of the specific chromosome abnormalities imparts a different clinical implication for the patient. In particular the presence of the t(94;14), t(14;16) and deletions of 17p13 are associated with a short survival. This knowledge is being used as a platform for drug development against this invariably fatal disorder.

ACKNOWLEDGEMENTS Rafael Fonseca is a Clinical Investigator of the Damon Runyon Cancer Research Fund. This work is supported by the International Waldenstro¨m Macroglobulinemia Foundation, and grants R01 CA83724-01, SPORE P50 CA100707-01and P01 CA62242 from the National Cancer Institute, and the Fund to Cure Myeloma.

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