Primary translocations appear to occur early during the course of MM, whereas secondary translocations occur later on and are involved in tumor progression. Most primary translocations are simple balanced translocations that juxtapose an oncogene and one of the Ig enhancers. An IGH rearrangement on 14q32.3 is found in most patients with MM. This rearrangement consists of complex and heterogeneous translocations with the breakpoint involving either the switch region of IGH or the V, D, or J gene. The primary translocations are caused by somatic hypermutation or errors in the V(D)J portion of the switch recombination region. The translocations include a promiscuous array of at least 20 nonrandom chromosomal partners.14q32 rearrangements are altered in 45% to 50% of MM patients. The characterization of these translocations has led to the identification of critical dysregulated oncogenes (e.g., BCL2, cyclin D). In each trans location, a potent enhancer is juxtaposed to dysregulated oncogenes. The five most frequent IGH translocations are t(4;14)(p16.3;q32.3), t(6;14)(p21;q32.3), t(11;14)(q13;q32.3), t(14;16)(q32.3;q23), and t(14;20)(q32.3;q12) (see Table 1).
Table1. Most Frequent Chromosomal Abnormalities in Multiple Myeloma and Their Frequencies
A recurrent CCND1-IGH rearrangement is characterized by over expression of cyclin D1. In contrast to mantle cell lymphoma (MCL), breakpoints on 11q13 with MM are not clustered but are scattered over a relatively large genomic region. Virtually all MM and MGUS cells have cyclin D dysregulation, suggesting that this abnormality is an early and unifying pathogenetic event. In contrast to other abnormalities involving the IGH locus, t(11;14) MM cells tend to be diploid. There appears to be an association of t(11;14) with an oligosecretory or light chain form of MM with CD20 expression, and a lymphoplasmacytic cell morphology. This chromosomal rearrangement is the most common genetic lesion in MM (15% to 20%) but it has also been observed in MGUS, primary plasma cell leukemia, and light chain amyloidosis. Two groups of patients with t(11;14) are recognized: one with a rather indolent course and the other associated with a more aggressive course.
Approximately 15% of patients have a recurrent t(4;14) (p16.3;q32.3) abnormality associated with aggressive disease. This cytogenetic abnormality is associated with IgAλ form of MM with immature cell morphology, and a poor response to therapy. This abnormality is cytogenetically cryptic and is detected by FISH. Two genes on chromosome 4p16.3 and the IGH switch region on 14q32.3 are involved. The fibroblast growth factor receptor 3 gene (FGFR3) is detected on der(14), where it is overexpressed along with a fusion of the MM set domain NSD2 (MMSET) gene, which is located on 4p16.3. This is the first example of a translocation that simultaneously deregulates two genes with oncogenic potential: the FGFR3 gene detected on der(14) and the NSD2 gene detected on der(4). FGFR3 is 50 to 100 kb telomeric to NSD2. Loss of FGFR3 on der(4) is detected in approximately 20% of cases. A significant number of these patients have del(1p32) and del(13q) within the same clone and are hypodiploid.
Translocation (14;16)(q32.3;q23) can only be detected with FISH studies. The incidence of t(14;16) has been estimated to be approximately 5% of cases. This translocation results in the relocation of the MAF proto-oncogene from its position on 16q23 to chromosome 14, band q32.3, and it is overexpressed. Survival of patients with t(14;16) is significantly shorter as compared with patients without t(14;16).
t(6;14)(p21;q32) is a rare translocation and present in approximately 1% to 2% of patients with MM. This rearrangement results in juxtaposition of CCND3 to the IGH enhancer and causes direct upregulation of CCND3 expression. The breakpoints are all located in the 3′ end of CCND3. The prognostic implication is neutral. Of the five recurrent translocations in MM the rarest translocation is t(14;20)(q32.3;q12)/IGH-MAFB, present in approximately 1%, and also results in the upregulation of MAFB gene. MAFB is also mutated in about 25% of patients harboring t(14;20). These MM patients have a poor prognosis, however, when t(14;20)(q32.3;q12)/IGH MAFB is detected in the MGUS or in smoldering MM stage of dis ease, it is associated with long-term stable disease.
A large number of secondary chromosomal aberrations are found during disease progression. Disease progression is accompanied by four main abnormalities including translocations of MYC, the loss or deletion of chromosome 13, deletions of short arms and amplification of long arms of chromosome 1, as well as the deletion of short arms of chromosome 17. Translocations and/or amplification of MYC (8q24) may involve up to 45% of patients with an advanced MM, and then cytogenetic studies frequently involve very complex nonreciprocal rearrangements, duplications, and amplifications. More recent data based on SNP-array technologies has revealed homozygous deletions such as BIRC2/3 on chromosome 11, deletions of TRAF3 on chromosome 14, and deletions of CYLD on chromosome 16. Another recurrent double deletion that has been observed in a limited number of the patients occurs on chromosome 1, targeting the tumor suppressor CDKN2C gene at 1q21 chromosomal site.
Deletion of chromosome 13 is detected in 10% to 20% of patients using conventional cytogenetics and in 50% using interphase FISH. The minimally deleted region on chromosome 13, between bands q14.11 to q14.3 contains over 68 genes including RB1, DNA marker D13S319, and micro RNAs mir16. -1 and mir15). Loss of the entire chromosome 13 (82%) is more frequent than is deletion of the long arms of chromosome 13. The median percentage of plasma cells carrying del(13q), as identified by FISH, ranges from 75% to 90%. Deletion of 13q is associated with specific clinical-pathologic features, including a higher frequency of λ-type MM, high plasma cell-labeling index, female predominance, and inferior survival after standard chemotherapy. t del(13q), however, no longer has adverse prognostic significance in patients treated with bortezomib. Molecular cytogenetics analyses have revealed that del(13q) is present in 90% of patients with t(4;14) or t(14;16); therefore the apparent adverse impact of del(13q) may be related to translocation events.
Deletion of TP53 at 17p13.1, which occurs in 15% of patients with MM, is a powerful independent predictor of shortened survival and is associated with clonal evolution, drug resistance, and genetic instability This deletion is not found in patients with other high-risk abnormalities such as t(4;14) or t(14;16), and it appears to be mutually exclusive. Patients with TP53 gene deletion have a significantly shorter OS regardless of therapy. The 17p deletion, identified by FISH, is considered the most important molecular cytogenetic fac tor when determining prognosis. In newly diagnosed MM patients del(17p) is observed in 8% of patients. Biallelic alteration of TP53 (deletion and mutation, a.k.a. “double hit”) is present in 3.7% of newly diagnosed MM and is associated with very poor outcome. Biallelic inactivation of TP53 has been reported to occur more frequently at relapse, having been reported at 21% to 26% of patients. Another study provides the most compelling evidence that del(17p) is an important prognostic factor even in the absence of TP53 mutation and should remain a high-risk feature in MM patients.
The gain of 1q21 locus has been identified in 45% of cases of MGUS, about a third of newly diagnosed MM, and 72% of relapsed MM, and represents one of the most frequent recurrent chromosomal abnormalities in MM (see Fig. 1). Chromosome 1 abnormalities include both short and long arms of chromosome 1, gains, deletions or jumping translocations. Gain of 1q is observed cytogenetically as an isochromosome, duplications, or jumping translocations and is detected with a 1q21-specific FISH probe. Among 479 patients with newly diagnosed MM, 43% with amp lq21 with either a hypodiploid and hyperdiploid karyotype and del(13q) was associated with poor prognosis as compared with patients lacking amp1q21 (see Fig.1). In a study of 92 patients treated with lenalidomide and dexamethasone, del(17p) and gain of 1q21 was associated with a dismal OS. In the recent retrospective study of 3578 patients with MM 24% had documented chromosome 1 abnormalities or high-risk chromosomal abnormalities within 90 days of diagnosis. Median OS was lower for patients with chromosome 1 abnormalities and these abnormalities were independently associated with an inferior OS, and were older and had high-risk cytogenetic abnormalities despite the use of state-of-the-art therapies. Similar observations were obtained when 1376 patients were examined at diagnosis for the presence of gains of 1q only; FISH analysis confirmed that 28% of patients with +1q, along with other high-risk chromosome abnormalities as well as chromosome 13 abnormalities had decreased OS.
Fig1. REPRESENTATIVE PARTIAL KARYOTYPES OF METAPHASE CHROMOSOMES DEMONSTRATING THE DIFFERENT TYPES AND DEGREE OF AMPLIFICATION OF CHROMOSOME 1. FISH probes for 1q12 (red), 1q21 (green), and 16q11 (aqua) are shown on inverted DAPI images of chromosomes. (A) Interstitial deletion of 1p (arrow) in the homolog on the left and a direct dup1q12–q23 on chromosome on the right. (B) Normal homolog 1 on the left and the abnormal homolog on the right, demonstrating both an interstitial deletion of 1p and the amplification of 1q in the same chromosome. Note four copies of 1q21 (arrows) in an inverted duplication pattern. (C) Examples of an unbalanced whole-arm translocation of 1q to chromosome 16q. Chromosomes 1 are on the left, and chromosomes 16 are 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) Examples 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) Examples of jumping 1q, in which all or a part of 1q is translocated to three 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 patient in (D). The der(21) results from the segmental translocation of the inverted dup of 1q to the short arm of 21. The der(22) results from the whole-arm 1q translocated to the short arm of 22. (F) Homologous of chromosome 1 demonstrating amplification of 1q12–q23 by breakage-fusion-bridge cycles. Note multiple copies of 1q21 (arrows) on the abnormal homologue on the right. The copies of the 1q12–q23 amplicon occur in an inverted repeated 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 1q12–q23 region by break-fusion-bridge cycles. FISH, fluorescence in situ hybridization. (Reprinted from Sawyer JR. The prognostic significance of cytogenetics and molecular profiling in multiple myeloma. Cancer Genet. 2011;204:3, with permission.)
In the most comprehensive expression profiling survey of patients with MM, reported by the Arkansas Multiple Myeloma Group using GEP70 (gene expression profiling of 70 genes), 30% of these genes were located on chromosome 1, with most of the downregulated genes located on the short arms of chromosome 1 and most of the upregulated genes on 1q. The mechanism for the amplification of 1q is believed to involve 1q12 pericentromeric instability, which most commonly increases the copy number of 1q by a direct and/or inverted duplication. Further instability can result in adding a whole arm segment of 1q to nonhomologous chromosomes by jumping translocations of 1q. The 1q12–q23 amplicon has been reported to contain the 1q12 pericentromeric heterochromatin and the adjacent bands of 1q, resulting in an inverted repeat pattern of amplification of the 1q12–q23 region. Copies of the 1q12–q23 amplicon can become integrated into complex multichromosome translocations during tumor progression. In vitro studies have shown that treatment with hypomethylating agents apparently amplify any 1q region juxtaposed to 1q12 chromosome band producing copy number aberrations in the bone marrow of these patients. Frequent additional deletions detected by conventional cytogenetics include regions within 6q, 8p, 12p, 14q, 16q, or 20p.
The median number of mutations per MM genome is about 55 to 60, with a very large range (21 to 488). Moreover, in contrast to leukemia, “good-risk” cytogenomic abnormalities have not been described. The current data indicate that genetic abnormalities have a major role in determining a patient’s prognosis. The current thinking is that development of subclones is a very early event in MM, prob ably occurring soon after the cell undergoes transformation.
Frequent mutations in MM include KRAS (particularly in previously treated patients), NRAS, BRAF, FAM46C (hyperdiploid subgroup), TP53, and DIS3 (in nonhyperdiploid MM with IGH rearrangement, TRAF3, CYLD, RB1, and PRDM1. Subclonal KRAS, NRAS, and BRAF mutations are observed in about one-third of patients with MM. The most recent exome sequencing study identified germline variant in DIS3 gene in 4 unrelated families with familial MM. The DIS3 gene, located on chromosome 13, band q22.1, and as mentioned above, chromosome 13 is recurrently deleted and somatically mutated in MM. This is the first description of germ line variant with potential involvement in MM etiology and extend beyond somatic alterations to germline susceptibility. The frequency reported is about 2.6% among four families with multiple cases of MM and MGUS.
Finally it’s important to mention that until recently, classification of MM patients and risk stratification has relied on either biochemical parameters, cytogenetic markers, or gene expression analysis. Novel MM-PSN, the first Patient Similarity Network (PSN) of newly diagnosed patients has enabled the identification of 3 broad patient groups and 12 distinct sub-groups defined by 5 data types generated from genomic and transcriptomic patient profiling of 655 patients.21 PSNs have been proposed as a potential powerful integrative approach to combine different omics data, to create a comprehensive view of a given disease and identify disease subtypes. The MM-PSN classification uncovered novel associations between distinct MM hallmarks with significant prognostic implications and allowed further refinement of risk stratification. Most important, MM-PSN confirmed that gain of 1q is the most important single lesion conferring high risk of relapse, and its association with an MMSET translocation as the most significant determinant of poor outcome. These findings suggest, as previously demonstrated in many studies, that gain of 1q should be incorporated in routine staging systems and risk assessment tools.
Combining novel single cell methodologies, such as sc RNA seq and sci CNV, a tool for inferring CNV from sc RNAseq allows dual profiling of DNA and RNA in a single cell. When MM cells with +1q were evaluated, using this dual profiling, it was recently uncovered that +1q cells in MM cause multiple effects on MM cells including a reduction in the unfolded protein response, enhanced oncogenic growth, oxidative phosphorylation, and MCL gene increased expression (anti-apoptotic gene located at 1q21.2). In contrast, there was no increase in CKS1B gene expression (located at 1q21.3) indicating that an alternative mechanism likely drives cell cycling in MM cells with a gain of 1q. Another gene, inflammation-responsive RNA editase gene adenosine deaminase acting on RNA1(ADAR1), also located at 1q21, has been recently implicated in the possible mechanism underlying 1q21 formation. ADAR1 is an RNA-editing enzyme that edits adenosine-to-inosine (A-to-I) nucleotides within double stranded RNA loops formed by primate-specific Alu repeats. ADAR1 has emerged as a vital contributor to transcriptomic diversity and is abundantly expressed in MM suggesting that MM cells with +1q transcriptome are in hyperediting state. In vitro and other functional studies suggests that ADAR1 can enhance the growth and proliferation of MM cells in an editing-dependent manner and sometimes independently of 1q amplification status implying that a disturbed transcriptome mediated by ADAR1 overexpression is both clinically and functionally crucial in myeloma. The interplay between the gain of 1q, IL6R (localized also on 1q21.3), and ADAR1 provides yet another possible mechanism for the adverse outcome of patients with MM and 1q abnormalities.
