Genetics in Myeloma: Genetic Technologies and Their Application

British Medical Bulletin, 2015, 113:15–30 doi: 10.1093/bmb/ldu041 Advance Access Publication Date: 6 February 2015

Genetics in myeloma: genetic technologies

and their application to screening approaches Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 in myeloma Polly J. Talley†,*, Andrew D. Chantry‡, and Clive H. Buckle‡

†Sheffield Diagnostic Genetics Service, Sheffield Children’s NHS Foundation Trust, Western Bank, Sheffield S10 2TH, UK, and ‡Sheffield Myeloma Research Team (SMaRT), Department of Oncology, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK

*Correspondence address. E-mail: [email protected]

Accepted 19 December 2014

Abstract Background: Despite advances in the treatment of multiple myeloma (MM), it remains an incurable malignant disease. Myeloma genetics is intrinsically complex, but it offers an opportunity to categorize the disease and apply a personalized medicine approach. Areas of agreement: Research into the genetics of myeloma is moving at a fast pace and is highlighting areas and patient cohorts likely to beneﬁt from speciﬁc treatment. Targeting residual disease is likely to be crucial to improved clinical outcome. Areas of controversy: Patients in clinical trials are more likely to receive genetic diagnosis than non-trial patients, for whom access is ad hoc and dependent upon regional commissioning arrangements. Areas timely for developing research: Relating genetics to potential treatment pathways will become crucial for improved myeloma outcomes. Universal access to standardized genetic testing will facilitate modern personalized treatments. Key words: myeloma, diagnosis, personalized medicine, genetic technologies

Introduction the bone marrow (BM) and overproduction of cir- Multiple myeloma (also known as plasma cell culating monoclonal immunoglobulin (paraprotein). myeloma) is a neoplastic disorder characterized by an The paraprotein product circulates in blood and abnormal monoclonal proliferation of plasma cells in is deposited in various tissues including the renal

© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] 16 P. J. Talley et al., 2015, Vol. 113 tubules, causing substantial renal impairment, and in 65 and 74 at diagnosis, and 37% are over 75. MM is other organs including the heart and the gut. At these diagnosed in younger people, with 37% of patients sites, the paraprotein is prone to transformation into aged 65 or under, and 2% below 40 years of age.5 a degradation-resistant protein known as amyloid, MM is not a disease that is seen in children.1 which significantly impairs organ function. Malig- Myeloma is likely to increase in incidence in the UK nant plasma cells secrete osteoclast-activating factors in line with the ageing population. Data suggest that and osteoblast-deactivating factors leading to destruc- MM is more common in men than in women (ratio tive, osteolytic bone disease. Malignant plasma cells 7.1:4.3), and more common in Black people than in accumulate within the BM effectively crowding out people of Asian or Caucasian descent (ratio ∼2:1).1,5 Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 normal haemopoietic tissue leading to BM failure There is a 3.7-fold increased risk to first-degree manifesting as anaemia, thrombocytopaenia and leu- family members of patients with MM, suggesting kopaenia as well as impaired cellular and antibody- some inherited component.1 mediated immunity. Fifteen per cent of MM patients have no symptoms Plasma cell neoplasms progress through distinct at presentation, and a high paraprotein may be discov- clinical phases: monoclonal gammopathy of unknown ered following routine screening. Approximately 40% significance (MGUS), asymptomatic myeloma or present with more substantial morbidity including smouldering myeloma (SM), plasma cell myeloma, anaemia, renal failure and skeletal disease including progressing to plasma cell leukaemia. Other clinical pathological fractures, hypercalcaemia, spinal cord phases following treatment can include myeloma in compression or generalized bone loss (osteoporosis). remission (termed ‘plateau phase’), relapsed myeloma Less critical symptoms can include backache, bone and refractory myeloma (where disease is non- pain, anaemia and tiredness. Diagnosis is dependant responsive to treatment). Plasma cell myeloma is on results from a number of clinical tests, including full defined by the presence of >10% clonal plasma cells in blood count and chemical analysis, serum and urine the BM, a paraprotein and the presence of end-organ electrophoresis, BM morphology, radiography and damage,1,2 which can be summarized by the acronym, genetic analysis. CRAB. Treatment strategies are conventionally divided into intensive and non-intensive regimens. The former • HyperCalcaemia feature chemotherapy using a combination of steroids • Renal insufficiency (e.g. dexamethasone), alkylating agents (e.g. cyclo- • Anaemia phosphamide) and immunomodulatory agents (e.g. • Bone lesions thalidomide or lenalidomide) plus or minus a prote- Hypercalcaemia results from bone destruction and is asome inhibitor (e.g. bortezomib or carfilzomib) for 3 fi seen in 20% of patients at diagnosis. Renal insuf - younger (<60 years), fitter patients, followed by ciency and ultimate failure is due to tubular damage autologous stem cell transplantation. High-dose mel- ∼ resulting from proteinuria. Anaemia is seen in 67% phalan (i.e. 200 mg/m2) is used as a consolidation of patients, and results from effacement of BM by therapy prior to autologous stem cell transplantation, plasma cells and renal damage resulting in loss of further reducing plasma cell load within the BM. erythropoietin. Osteolytic disease is seen in 70% of Non-intensive regimens comprise similar combina- 1,4 patients with MM. tions used at attenuated doses and without autolo- Myeloma is the 17th most common cancer in the gous stem cell transplantation.4,6 UK accounting for 1.5% of all new cases reported Immunomodulatory drugs, such as lenalidomide (source: Cancer Research UK, 2009). This equates to (and other thalidomide analogues), have been shown ∼5000 new patients being diagnosed with myeloma to have clinical efficacy in the treatment of myeloma. in the UK per year. MM is a cancer of older adults: Recent work has shown that these immunomodu- the majority of cases occur in patients over the age of latory drugs can bind to and inhibit the cereblon 65. Twenty-six per cent of patients are aged between ubiquitin ligase. Furthermore, lenalidomide-bound Review of genetic screening in myeloma, 2015, Vol. 113 17 cereblon acquires the ability to target two specific immunoglobulin (Ig) antibody molecule is composed lymphoid transcription factors, IKZF1 and IZKF3, oftwoheavychainandtwolightchainproteins.These both known to play a central role in B and T cell are encoded by the IGH gene for the heavy chain, biology, for selective ubiquitination and degrad- located on chromosome 14, and the IGK and IGL ation.7,8 Proteasome inhibitors are also extremely genes for the light chain, located on chromosomes 2 effective in myeloma treatment. The proteasome is an and 22, respectively.15 Variable gene segments at these intracellular enzyme complex that breaks down loci undergo irreversible rearrangement, at the DNA damaged proteins. The exquisite sensitivity of myeloma level, and this creates individual B cells with specificity to proteasome inhibitors remains largely unexplained, for a single antigen. In summary, the several stages of Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 although the drugs are thought to act in a multifaceted B-cell development each represent a change to the and extensive mechanistic fashion. They have been genomic DNA involving the variable (V), diversity (D) thought to stimulate apoptotic pathways, inhibit the and junctional (J) gene segments of the immunoglobu- NF-KB pathway, down-regulate expression of genes lin genes. These stages can be divided into three pro- associated with DNA repair and induce an endoplasmic cesses, all of which generate double-stranded DNA – reticulum stress response.9 11 Bisphosphonates are an breaks: VDJ recombination, somatic hypermutation important class of drugs used to treat bone manifesta- and IgH-switch recombination.17,18 tions in MM. They are potent inducers of osteoclast Following maturation, plasma cells have under- apoptosis, thereby reducing elevated bone resorption gone the final stages of development and home to the associated with MM. BM.17 These cells are long-lived, terminally differen- Shorter survival times correlate with higher clinical tiated and non-dividing. They are highly dependent stage at diagnosis, renal insufficiency, degree of on the BM microenvironment where their survival is marrow replacement, increased proliferative activity favoured by survival factors found in permissive and certain karyotypic abnormalities.12 Although sig- niches.19,20 nificant advances have been made in MM treatment Plasma cells interact with the BM microenviron- over the past two decades, and it is now regarded as ment via a number of complex interactions, which are highly treatable, myeloma remains almost always crucial to tumour survival and disease progression.19 incurable. The only wholly curative option available The BM microenvironment is made up of extracellul- for the treatment of myeloma is allogeneic stem lar matrix and five types of BM stromal cells: fibro- cell transplantation. However, although this intensive blastic stromal cells, osteoblasts, osteoclasts, vascular procedure can achieve long-term remission, it is asso- endothelial cells and lymphocytes.21,22 The crosstalk ciated with a high-treatment related mortality and between myeloma cells and the microenvironment risk of relapse and tends to be considered only in a can occur either directly or indirectly, via local secre- small minority of patients.13 Eighty-eight per cent of tion of adhesion molecules, cytokines and growth myeloma patients will survive 1 year, median survival factors. The strong association between these cell is 3 years and 10-year survival is only achieved in types is thought to be pivotal in the pathogenesis 10% of patients.12 Importantly, there is a subset of of osteolytic lesions and bone loss associated with patients for whom current treatment modalities are MM.17 In both MGUS and early MM, plasma not effective, with ∼20% of clinical responses sub- cells are highly stromal dependant; this dependence optimal and 5% non-responsive. decreases as the disease progresses. Many of the cellular interactions involved are governed by genetic changes affecting their normal regulation.19 Plasma cell biology While key to the creation of a diverse adaptive The human immune system has evolved to confer immune repertoire, these complex developmental resistance to infection. Plasma cells are part of the processes imply an inherent genome instability, and ‘adaptive’ component and are required to produce it is this required instability that may facilitate onco- – antibodies in response to antigenic insult.14 16 The genic transformation. 18 P. J. Talley et al., 2015, Vol. 113

Genetics of plasma cell neoplasms gives rise to abnormal expression of the oncogene and contributes to the myelomagenic effect. Detailed Myeloma is a genetically complex disorder charac- in Table 1, the five main translocation partners are terized by multiple genetic changes, affecting differ- the following: t(4;14), t(11;14), t(6;14), t(14;16) and ent pathways, that have the ability to deregulate t(14;20); together these are seen in ∼40% of patients. plasma cell biology leading to a broadly similar These rearrangements are thought to be associated phenotypic manifestation of disease.19 This genetic with up-regulation of one of the cyclin D genes. heterogeneity, in part, is likely to have hindered the t(11;14) and t(6;14) directly deregulate CCND1 and development of effective treatments. A deeper and CCND3, respectively; t(14;16) up-regulates CCND2 Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 clearer understanding of the genetic abnormalities, as MAF directly binds to the CCND3 promoter and their role in specific pathways, may offer new andt(4;4)alsoup-regulatesCCND2 via FGFR3 and routes for drug development. This could enhance the MMSET, although the exact mechanism is unknown.19 clinicians’ ability to offer a personalized medicine Such translocations are mediated by errors in DNA approach when treating patients with MM. modification associated with B-cell maturation, following plasma cell–antigen interactions. IGH switching is Hyperdiploidy vs. hypodiploidy considered the most common, while somatic hypermutation and VDJ recombination are likely to be less From a genetic perspective, myeloma can be divided frequent events. into those with and without a hyperdiploid karyo- The t(4;14)(p16.3;q32) translocation is cytogen- 21,23 ∼ – type. Hyperdiploidy is seen in 30 40% of etically cryptic.21 As a result, fluorescence in situ patients. Chromosome number ranges from 48 to hybridization (FISH) is required to determine the pres- 75, median 53, and the chromosome gains are non- ence of this rearrangement. The t(4;14) rearrange- random, often involving the odd numbered chromo- ment involves the IGH gene and two protein-coding 24 somes, 3, 5, 7, 9, 11, 15, 19 and 21. These changes genes located at 4p16.3, multiple myeloma SET are not seen as serial gains, but rather as the result of domain (MMSET) and the fibroblast growth factor 19 a single catastrophic mitotic event. Hyperdiploidy receptor 3 (FGFR3), an oncogenic receptor tyrosine in myeloma has been associated with a better overall kinase.21,28,29 In the balanced translocation, both survival when compared with the non-hyperdiploid FGFR3 and MMSET are juxtaposed to two IGH 25 group. enhancers. FGFR3 is overexpressed from the deriva- The non-hyperdiploid group includes karyotypes tive chromosome 14, and MMSET is overexpressed with hypodiploid, pseudodiploid or near tetraploid from the derivative chromosome 4. Twenty-five per 25 chromosome number. The near tetraploid groups cent of cases are seen in an unbalanced form, with appear to be a doubling (i.e. 4n) of the hypodiploid loss of the derivative chromosome 14, which is asso- and pseudodiploid cell lines. The non-hyperdiploid ciated with the loss of the aberrant FGFR3 expres- groups are typically associated with IGH transloca- sion.21,23 This translocation is universally associated tions, although IGH rearrangements are also present with a poor prognosis. It is seen in MGUS (∼3% of ∼ 21 in 10% of the hyperdiploid group. cases), but more highly associated with SM and – MM.30 32 Data suggest that these patients benefit from bortezomib therapy, and in addition, TKI-258, Translocations an FGFR3 inhibitor, is also undergoing clinical Non-hyperdiploid patients are frequently (55–70%) evaluation.23 associated with rearrangements of IGH on chromo- t(11;14)(q13;q32) involves translocation of the some 14.19,26,27 IGH rearrangements are considered CCND1 gene located at 11q13, where it comes promiscuous as they have many partner genes. They under the regulatory influence of IGH at 14q32.33 are usually simple reciprocal translocations juxta- This translocation is balanced in the majority of posing the IGH enhancers to an oncogene.21 This cases, although additional copies of the derivative Review of genetic screening in myeloma, 2015, Vol. 113 19

Table 1 Detailing the ﬁve main translocations associated with IGH in myeloma and the normal gene role or function

Translocation Gene(s) Frequency Prognosis Gene role/function t(4;14)(p16.3;q32) FGFR3 15% Poor Bone development and maintenance MMSET Oncogene, overexpression results in proliferation and anti-apoptotic effects t(11;14)(q13;q32) CCND1 15% Good Cell cycle G1/S transition

t(14;16)(q32;q23) MAF 5% Poor Oncogene, enhances tumour stroma interactions Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 t(6;14)(p21;q32) CCND3 3% Good Cell cycle G1/S transition t(14;20)(q32;q11) MAFB 2% Poor Regulation of lineage-speciﬁc haematopoiesis

chromosome 14 have been seen and are thought to events involving more than one chromosome and be associated with progressive disease. associated regions of amplification and duplication The t(6;14)(p21;q32) involves the gene CCND3 (Fig. 1C).21 The t(8;14)(q24;q32) accounts for only at 6p21, resulting in its up-regulation. It is often seen 25% of MYC rearrangement,17,36 and recent studies on a backdrop of a complex karyotype, and the have demonstrated that MYC is able to recruit active derivative chromosome 14 can be present in multiple super enhancers from highly expressed genes asso- copies. This is a rare event and has only been associated with B cell, plasma cell or myeloma develop- ciated with ∼3% of MM patients.19 ment.37 Examples include enhancers associated with t(14;16)(q32;q23) and t(14;20)(q32;q11) both show CCND1, XBP1, KRAS, FAM46C and CHST15.37 juxtaposition of the IGH gene to a MAF family gene, MAF and MAFB, respectively.34 MAF rearrangements have been described in 5–7% of MM and considered to Deletions and duplications be mediated by a fragile site in the WWOX gene on MM genetics includes copy number variation as a the long arm of chromosome 16.17,23 This results in common and recurrent finding.19,23 Theoretically, MAF coming under the regulatory influence of IGH, and simplistically, deletions are likely to involve loss whichinturnhastheeffectofup-regulatingCCND2.19 of tumour suppressor genes at those sites, and dupli- MAF rearrangements have been associated with a more cations are likely to be associated with overexpres- aggressive clinical course.23 Data on MAFB rearrange- sion of genes within the region. Regions of recurrent ments associated with t(14;20) (Fig. 1A and B) are not deletion and/or duplication include 1p, 1q, 9q, 11q, robust due to their rarity, although a similar clinical 12p, 13q, 15q, 16q, 17p, 19q and 22q,17,19,23,38 and course would be predicted.23 a number of these are described further. Translocations not involving IGH do occur, Deletion of chromosome 13 was the first chromo- but they are considered unusual and are most likely some abnormality to be associated with a poor prog- to be seen in progressive disease. CCND3-MAF, nosis in myeloma.39 Chromosome 13 abnormalities MAF-FGFR3/MMSET, CCND3-FGFR3/MMSET re- are seen in 50% of myeloma patients;23 of these, arrangements have all been described.19 85% show chromosome 13 monosomy, and the MYC translocations are seen in ∼15% of patients remaining 15% involve an interstitial deletion of with myeloma at presentation21,35,36 and upto 45% 13q14 which includes the RB1 gene.36,40 Recently, of patients with advanced disease.21,23 MYC translo- the poor prognosis associated with deletion/mono- cations are not considered to be initiating events, but somy of chromosome 13 has been contested in the rather late events associated with increased prolifer- literature. Its close association with the presence of ation and stromal independent plasma cells.21 They t(4;14) suggests that the statistically poor prognosis are frequently seen as non-reciprocal translocation may be a result of association alone.23,41 20 P. J. Talley et al., 2015, Vol. 113 Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021

Fig. 1 Partial karyotypes and FISH images of genetic abnormalities associated with MM. (A and B) The partial karyotype shows the normal chromosome 14 and the normal chromosome 20 on the left of each pair. Additional material can be seen on the long arm of the derivative chromosome 14, from the derivative chromosome 20 depicting the t(14;20)(q32;q11). The FISH image using the Cytocell probe set for IGH and MAFB conﬁrms that the rearrangement involves the juxtaposition of IGH and MAFB.(C) FISH image depicting the Vysis MYC break apart probe. There is an intact copy of the probe on one chromosome 8. The second fusion has broken apart but also demonstrates associated complexity with duplication of the centromeric part of the probe and involvement of three chromosomes. (D and E) The partial karyotype shows a normal chromosome 1 on the left with an isochromosome 1q on the right. The isochromosome shows two copies of the long arm mirror imaged around the centromere with effective loss of the short arm of chromosome 1. The FISH image shows the CytocellCDKN2C/CKS1B probe that highlights a gene of interest on the long arm in red and a gene of interest on the short arm of chromosome 1. A three red and one green signal pattern conﬁrms the gain of 1q and the loss of 1p. Coloured arrows demonstrate the probe positions, green and red arrows show green and red signals, respectively; yellow arrows depict fusion signals.

Deletion of the short arm of chromosome 17, the that have been associated with this deletion include site of the TP53 tumour suppressor gene, remains CDKN2C, FAF1 and FAM46C.19,44 Rearrange- the most important prognostic factor in myeloma gen- ments of the long arm of chromosome 1 are often etics19,23 conferring an extremely poor prognosis.42 complex21,45 and have been associated with peri- Deletion of 17p has been associated with shorter sur- centromeric instability. They are seen in 40% of vival, aggressive disease, increased hypercalcaemia myeloma patients at diagnosis and 70% of cases at and the presence of extramedullary disease.23 relapse.21 ‘Jumping’ translocations are frequently Chromosome 1 rearrangements are the most associated with chromosome 1q duplication,21 and common aberrations in myeloma.21 These usually this term describes rare chromosomal events in result in deletions of the short arm and duplications which the same donor chromosome segment is trans- of the long arm.21,23 Short arm deletions have been located onto two or more recipient chromosome shown to span a region from 1p13 to 1p31 and have sites, such that it can be present in different guises in been associated with a poor prognosis.21,43,44 Genes different cells. Implicated genes include CKS1B and Review of genetic screening in myeloma, 2015, Vol. 113 21

ANP32E.21 Duplication of 1q is also associated with is thought to have an anti-apoptotic effect.19 In add- poor prognosis, although the intrinsic relationship ition, data suggest that low-level TRAF3 expression with deletion 1p creates difficulties in assessing these is associated with a better response to bortezomib.23 abnormalities separately.21 A common manifestation Other affected pathways include the Wnt signal- of del1p/dup1q is the isochromosome 1q, (Fig. 1D ling pathway, RANK, PI3K, JAK and the mitogen- and E), where the short arm is lost and the long arm activated protein kinase (MAPK) pathways. Bone is duplicated and mirrored around the centromere. damage associated with MM results from an im- Uniparental disomy (UPD) represents a further balance in osteoblast and osteoclast function. The mechanism by which alleles can be lost. UPD is a Wnt signalling pathway supports osteoblast function; Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 term used to describe loss of a chromosome, or therefore, pathway inhibition results in osteoblast chromosome part, which is then duplicated from the suppression and reduced bone formation. The RANK second allele. This results in loss of heterozygosity pathway is often enhanced in myeloma, and the (LOH) without the associated loss of copy number. resulting MIP1-α activation stimulates osteoclasts, UPD can be seen associated with cancer genotypes resulting in increased bone resorption.4,49 The MAPK and is termed ‘acquired UPD’. Walker et al.46 have pathway is dysregulated in ∼55%ofMMpatients. demonstrated that acquired UPD/LOH is prevalent Genes that have been associated with MAPK signal- in MM with a median number of three UPD regions ling include KRAS, NRAS, BRAF and more recently per sample. The size ranged from 677 kb to whole NF1 and RASA2.50 chromosomes, but tended to implicate relatively small regions at 1p, 1q, 6q, 8p, 13q and 16q. These areas of UPD may highlight areas containing genes Epigenetic changes important in myeloma pathology. Epigenetic factors are also involved in the aetiology of MM, with changes to both DNA glycosylation/acetyl- Mutations and implicated pathways ation and histone modification playing a part in modu- lating gene expression. Global DNA hypomethylation Recent studies involving whole exome sequencing and specific gene hypermethylation have been reported (WES) have suggested that an average of 35 genetic in association with the transformation of MGUS to mutations is present per MM patient sample. This MM.19 Fifteen per cent of t(4;14) patients show a gene- figure sits part way between the suggested mutation specific hypermethylation pattern. Overexpression of level of 8 in the more genetically simple haemato- MMSET leads to histone modificationwhichinturn logical malignancies and ∼540 mutations detected in promotes cell survival and cell cycle progression.19 Dys- genetically complex epithelial tumours.47 regulation of miRNA has been associated with a par- Determination of these multiple mutations, and the ticular gene cluster on chromosome 13 implicated in the general genetic heterogeneity, supports the hypothesis MGUS to MM transition.51 that a pathological requirement of MM is pathway deregulation, rather than specific gene rearrangement or modification. Do the multiple genetic abnormalities converge to result in a more simplified effect, of target- Genetic predisposition ing a smaller number of specific functional pathways? An inherited genetic variation at 2p23.3, 3p22.1 and Keats et al.48 have demonstrated constitutive acti- 7p15.3 has been associated with a genetic predispos- 52,53 vation of the NF-KB pathway in 50% of MM cases ition to MGUS. More recently, genome-wide asso- using gene expression profiling. This is not caused by ciation studies (GWAS) have shown further regions of a single mutation, but rather a collection of gene common variation at 3q26.2, 6p21.33, 17p11.2 and mutations and deletions. Genes implicated include 22q13.1.54 Chubb et al.54 suggest that the seven loci BIRC2 and BIRC3 at 11q, CYLD at 16q and identified are likely to account for ∼13% of the famil- TRAF3 at 14q32. Increased NF-KB nuclear activity ial risk of myeloma, and this highlights the likelihood 22 P. J. Talley et al., 2015, Vol. 113 that many more variants associated with a genetic sus- formation of the spindle apparatus and thereby induce ceptibility await discovery. cell-cycle arrest at the metaphase stage of mitosis. At metaphase, the chromosomes have condensed and are preparing to arrange themselves on the equator of the Intraclonal heterogeneity spindle apparatus, before anaphase proceeds to split Recently, WES, cytogenetics and copy number ana- the two chromatids into separate daughter cells. Har- lysis have been used to demonstrate that the majority vesting samples at this stage and treating chro- of myeloma cases have a complex subclonal structure. mosomes with trypsin and stain (e.g. Leishmans or Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 Bolli et al.50 have demonstrated, using serial sampling, Giemsa) produces a characteristic banding pattern, that clonal evolution is diverse and includes both allowing chromosomes to be karyotyped. linear and branching evolution. This suggests that Although conventional G-banded cytogenetic fi myeloma populations are not homogeneous, but analysis offers the bene t of a whole genome ana- made up of different populations with different clon- lysis, albeit at low resolution, the requirement for ally related and unrelated changes. These clones can cells in metaphase can be extremely problematic due demonstrate a differential response to therapy. This to the low proliferative rate of terminally differen- raises treatment issues; reducing the level of one clone tiated mature plasma cells. Even introducing adapta- may result in expansion of a more prognostically detri- tions to the culturing process, allowing for longer – mental clone. The process of branched evolution and term culturing (4 6 days), cytogenetic abnormalities ∼ 32 the existence of intra-tumour heterogeneity provide are only reported in 30% of myeloma patients. fl further ambiguity, since a single genetic picture at a This low-level detection is likely to re ect failure to single time point is likely to under-represent the com- capture the plasma cells in division, rather than a fl plexity of the underlying disease.55 re ection of the true abnormality rate. This is borne out by the much higher abnormality rate reported by FISH techniques. Genetic analysis in myeloma Cytogenetic analysis requires the individual kar- (advantages and disadvantages) yotyping of a number of cells, this time consuming, skilled analytical step contributes to the slow and Genetic analysis of BM samples in myeloma patients expensive nature of this methodology. has proved to be useful, offering both diagnostic and prognostic information to the clinician and the patient.4 Since myeloma is a genetically heteroge- neous disease, it is likely that genetic abnormalities FISH will increasingly be used to inform treatment deci- FISH involves hybridizing fluorescently labelled target 13,56 sions. Patients exhibiting a t(4;14) are more likely DNA, from a region of interest, to metaphase or inter- to be offered more aggressive therapy, including bor- phase cells to allow enumeration or rearrangement fi tezomib, rst line, and BRAF inhibitors incorporated detection (Fig. 2). The ability to utilize interphase cells where BRAF mutations exist. eliminates the problems associated with metaphase capture for cytogenetic analysis. FISH allows analysis of increased cell numbers Cytogenetic analysis (50–200 cells). However, this remains problematic Cytogenetic analysis in MM relies on the ability to since plasma cell populations can be as low as 10% capture plasma cells during the metaphase period of in MM patients and lower still in MGUS. Methods cell division. Robust techniques are employed in many of plasma cell enrichment are usually employed to laboratories to manipulate the cell cycle. These often overcome this issue. Plasma cells can be sorted using utilize colchicine, or one of its analogues, to disrupt magnetic-activated cell sorting (MACS™), which Review of genetic screening in myeloma, 2015, Vol. 113 23 Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021

Fig. 2 Schematic diagram to demonstrate the process of FISH. Probe DNA of interest, usually relating to a specific gene rearrangement or deletion, is labelled with a fluorescent dye and co-denatured alongside the target DNA to create single-stranded DNA. The single-stranded DNA is hybridized together allowing competitive reannealing to occur. A series of post-hybridization washes remove any excess probe and the target and probe can be visualised using a fluorescence microscope.

entails collection of CD138-positive cells or coF- DNA microarray/comparative genome ISHed using fluorescent cytoplasmic immuno- hybridization globulin (cIg) stain, to highlight plasma cells. An DNA microarrays employ similar technology to abnormality rate of 90% is reported in MM samples comparative genome hybridization (CGH). Both when FISH techniques are utilized. DNA of interest and control DNA are prepared and The resolution of FISH is limited by probe size, fluorescently labelled: control DNA in red and DNA which ranges from 150 kb to 1 Mb. Small deletions of interest in green. The two DNA samples competi- or mutations within genes or within the probe target tively hybridize to an array chip displaying comple- cannot be detected. Specific probes have an asso- mentary DNA oligonucleotides. In the case of equal ciated false-positive rate, dependant on the probe set copy numbers in the two DNA samples, the com- design. This rate is typically quoted at 1–4% by petitive hybridization will be equal. Increased copy manufacturers. number in the DNA of interest will result in a stron- FISH can be an expensive technique when mul- ger green fluorescence signal. Conversely, when dele- tiple probes are used, but small panels offer an effi- tions are present in the DNA of interest, reduced cient and accurate way to detect rearrangements and competition will result in dominant red fluorescence. copy number changes. Hyperdiploidy can be confi- The level of fluorescence is measured across the chip dently assessed using a 3-chromosome combination for each oligonucleotide to identify regions of loss of probes (chromosomes 9, 11, 15). The presence of and gain across the genome. two of the three chromosomes is a highly specific DNA microarrays are designed to provide a full indicator of hyperdiploidy.57 genome screen with a backbone level of resolution 24 P. J. Talley et al., 2015, Vol. 113 across the genome, and areas of specificinterest(for primer sequences present in each of the pairs. One example, cancer-related probe sets) can have enhanced member of the pair incorporates a stuffer sequence, resolution. Current arrays permit interrogation of the thus creating a known total probe length. The DNA entire genome using >2.6 million markers for copy is denatured and hybridized with the MLPA probes number analysis and ∼750 000 single nucleotide poly- that attach to the region of interest. When the DNA morphisms (SNPs). The higher probe density areas target is present with no alterations, the probe pairs include >500 cancer genes, all of which are covered lie immediately adjacent to each other and can ligate. with 25 markers per 100 kb region. Although SNP When there is a deletion, or in some cases a muta- incorporation allows for extended analysis of LOH, tion, the probes cannot hybridize to the DNA cor- Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 DNA array technology remains predominantly a high- rectly, and ligation does not occur. PCR is used to resolution method for detecting copy number changes directly amplify the ligated probe pairs and not the since balanced translocations and mutations at a base target sequence. As only ligated probes will be ex- pair level are not detectable. ponentially amplified, the number of products is a direct measure of the number of target sequences, compared with control. Separation of the products is Gene expression analysis carried out using electrophoresis, and the known Gene expression profiling (GEP) analysis takes the probe lengths allow identification of the specific analysis of genetic change one step further and target region; the steps are depicted in Figure 3. allows the impact of these changes on the messenger MLPA offers a cost-effective, multiplex technique RNA (mRNA) to be measured by assessing the activ- to assess loses and gains in up to 50 regions across the ity of the cells at a point in time. This is usually done genome. The limitations of MLPA are that, used in by simultaneous comparison between patient sample isolation, only copy number changes can be detected time points or against a normal control. DNA modi- (and only in the regions that have been targeted by fications in myeloma have been shown to be accur- the probe set). MLPA cannot distinguish between a ately assessed by GEP, offering a comprehensive and mutation and a deletion, as both scenarios result in sensitive approach for myeloma classification and non-amplification. The technique is highly sensitive to risk stratification.58 Issues relating to the complexity contamination, and furthermore, low-level abnormal of the analysis, interpretation and maintenance of populations can be problematic. In the case of MM, good quality control make this a less robust technique plasma cell purification to a minimum 50% purity in a routine clinical setting. The high risk groups, spe- would be a prerequisite. MLPA has proved its worth cifically, have a broad range of clinical outcomes, sug- in studies of MM; however, as its power is restricted gesting that further work is required to create robust to detection of gains and losses, it should be used in analytical methodologies before GEP can be utilized conjunction with FISH for detection of IGH rearran- routinely.58 gements.59

Multiplex ligation-dependant probe Next-generation sequencing fi ampli cation The emerging technologies of high-throughput or Multiplex ligation-dependant probe amplification next-generation sequencing (NGS), including whole (MLPA) is a multiplex polymerase chain reaction genome sequencing (WGS), WES and targeted NGS (PCR) methodology, which allows simultaneous panels, offer the highest level of resolution for genetic detection of deletions or duplications of up to 50 tar- screening. NGS has evolved dramatically over the last geted gene regions. As the probe sequence is small few years; it offers the power of simultaneous sequen- (∼60 nucleotides), it can detect deletions of an exon, cing of multiple DNA templates, without the require- smaller than conventional FISH techniques. MLPA ment of the same number of target-specificDNA probes are designed in pairs with the same two primers.60 Commercial platforms have also evolved Review of genetic screening in myeloma, 2015, Vol. 113 25 Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021

Fig. 3 Schematic diagram to demonstrate the process of MLPA. Paired probes around an area of interest are labelled with primer sequence. A stuffer sequence of known length is also included to allow for probe identification. The probes are annealed to the DNA of interest. When no DNA alteration is present, the probes lie adjacent to each other allowing probe ligation to occur. Ligation is not possible where deletions or mutations are present. Ligated probes are amplified and represent a measure of the number of copies. The products are separated by electrophoresis based on their size. providing confidence in the robustness of this app- specifically from the same clone, making the signals in roach to provide accurate and comprehensive means the later stages more easily readable. of mutational analysis. Emulsion PCR is based on the technique of pyrose- There are a range of NGS platforms currently on quencing in which the reaction is flooded sequentially the market, each of which employs different sequen- and repeatedly with each of the four deoxyribonu- cing chemistries but with similar data output. All cleotides, G, C, A and T. Incorporation of a nucleo- begin with the production of short DNA fragments tide results in emitted light, or a change in pH, which with the addition of non-specific adaptor DNA is captured, allowing the sequence to be accumulated sequences. This can be achieved either by chemical over hundreds of flow cycles. The Illumina™ chemis- binding or ligation.61 Adaptor sequences allow DNA try utilizes sequencing primers that are complementary fragments to be captured onto minute bead structures to the ligated adapter sequences. The PCR reaction or tethered to a solid surface (microchip). Subsequent mix is made up of chain terminating deoxyribonucleo- PCRisthencarriedoutinemulsionwithintinywells tides, labelled with a specific fluorescent tag, which (big enough to hold one bead) or by implementing a corresponds to the base identity. During the extension bridging PCR reaction. In all approaches, this step phase, termed sequencing by synthesis (SBS), each allows single DNA fragments to be amplified non- individual base is recorded by its fluorescence. These 26 P. J. Talley et al., 2015, Vol. 113

fluorescent tags are then cleaved, removing the chain specific function. Analysis is limited to the pre-selected terminating effect and allowing further extension to genes, and therefore, the functionality of novel gene occur and the sequence to build up sequentially. Once discovery is lost. Despite this, panels can be designed thesequencefragmentsarecreated,theyarealignedto with specific objectives in mind, for example tailored the known reference sequence using bioinformatic to the cohort of patients/samples to be analysed. In algorithms. The computational technology is able to thecaseofMM,wemaychoosetotargetthe measure the number of fragments over a given region ‘osteome’—genes implicated in the normal or patho- (read depth) and highlight (call) sites of discordance logical biology of bone. to the reference sequence (variance).60 There are pros To date, NGS has been applied successfully by the Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 and cons relating to each chemistry type, but modifica- haematology research community, but it is slower to tions and updates are fast coming and many of these translate to diagnostic services. Myeloma is included issues are being overcome with time.61,62 in this flurry of research activity. In 2011, Chapman NGS technology has superseded Sanger sequen- et al.47 applied both WGS and WES technologies to a cing, which has been the primary sequencing method cohort of 38 myeloma patients, and their data present for ∼30 years. Sequencing the human genome took novel mutations in genes associated with histone years to complete using Sanger technology. NGS, at methylation and protein translation, and in addition, its current best, reports production of 25 gigabases they provided additional evidence of the importance 61 per day—the equivalent of eight human genomes. of the NF-KB pathway, demonstrating mutations in What NGS can offer in the research setting, by way of 11 relevant genes. The study also reported BRAF novel findings and identification of mutations, could involvement in 4% of MM patients, raising the possi- be considered problematic when transitioning this bility of incorporating BRAF inhibition in treatment technology into the diagnostic forum. The expense, for these patients.64 WGS has also been assessed at the magnitude of data, time-consuming analysis and four time points across a single patient’s disease interpretation, limited analytical software currently course. The patient was shown to have t(4;14), seen available, limited theoretical understanding and issues at all time points, as well as 10 single-nucleotide var- of consent and ethics relating to incidental findings iants. Other genetic changes appeared and disap- (i.e. finding an abnormality in a gene unrelated to the peared throughout the disease. Five new events were specific investigation)63 all remain cause for concern. seen at the final time point of plasma cell leukaemia, Nonetheless, implementation of NGS technology in and these changes have been postulated as leukaemic diagnostic laboratories is moving forward apace. transformation events.65 There are number of ways of applying NGS tech- NGS remains relatively expensive, time consuming nology in a more manageable way, from a techno- and has the potential to generate excessively large logical, analytical and financial point of view. This data sets. More targeted approaches offer realistic often involves targeting smaller regions of interest— aims in terms of the diagnostic management of MM, WES. In this scenario, only the exonic, protein-coding since they retain the ability to determine the mutation gene regions are sequenced. This amounts to ∼1.5% status over a large number of relevant genes. Although of the whole genome, 30 megabases of DNA or adaptations are fast being included in the NGS reper- 180 000 exons, but is believed to contain 85% of toire, it is not designed to assess large regions of dupli- disease-causing mutations.61 cation or deletion, or balanced rearrangements. NGS panels offer a chance to harness the accuracy and depth of NGS analysis in an even more targeted fashion. This method has proved a popular way to Conclusions introduce NGS sequencing to the diagnostic setting. Despite advances in the treatment of MM, it remains A set of genes is selected to create a study panel. an almost invariably incurable malignant disease. Sequencing can be aimed at a particular chromosomal Myeloma genetics is intrinsically complex and hetero- region, genes implicated in a specific disease or a geneous, but it offers an opportunity to categorize the Review of genetic screening in myeloma, 2015, Vol. 113 27 disease into genetic subtypes. Myeloma genetic >10% plasma cells in the BM aspirate or trephine. research is highlighting areas and patient cohorts An age limit or fragility limit may be appropriate, where targeted treatments would be appropriate. As a meaning that patients not likely to be considered for result of this genetic, as well as clinical, complexity, it treatment may not benefit from genetic testing, is becoming clear that there is unlikely to be a single although there is a strong argument that the prognos- approach to treatment of myeloma patients, but tic information is useful in patient management even rather a series of genetically targeted and systems tar- if hard line treatment is not an option. It is generally geted treatments that are likely to be used in com- accepted that genetic testing should be carried out on bination to create an orthogonal cancer therapy selected plasma cell populations. The abnormalities Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 approach based on the genetic subtypes of MM.66 targeted are likely to include those with a prognostic The complex genetic findings in MM mean that a impact; IGH rearrangement (and its partners), TP53 single genetic technology cannot currently provide deletion, 1p and 1q abnormality, but should perhaps information across the breadth of genetic diversity be extended to include hyperdiploidy, or those abnor- encountered. Cytogenetic analysis is expensive and malities that already have inhibitors that are poten- only offers clinically significant information in a tially available, for example BRAF rearrangement. fraction of patients; FISH is an accurate and highly Then the question of technology arises; it may be that applicable test, but it becomes costly and time con- this is not the most important factor. While poten- suming as increased numbers of probes are utilized; tially NGS could offer the flexibility of analysis of a MLPA and array technology offer the higher reso- whole range of abnormalities, its expense precludes it lution and the ability to target multiple lesions, but it currently. A single test does not yet cover the range of cannot detect balanced translocations or rearrange- appropriate testing, but a comprehensive FISH/MLPA ments; GEP has the ability to risk stratify patients, regime or FISH/array regime could be envisaged at a but it is not currently able to determine all areas of cost that is likely to be considered appropriate within prognostically useful information. NGS is a novel the context of myeloma diagnosis and treatment. Best technology, and although well used in the research practice guidelines would not only provide uni- setting, it is not yet incorporated into all NHS diag- formity, but also offer the most comprehensive infor- nostic centres. Importantly, this ‘high-throughput’ mation possible to clinicians and therefore the best approach has the potential flexibility to create a treatment options for patients with MM. testing strategy aimed to cover all known prognostically relevant MM aberrations. Funding Targeting residual disease is likely to be crucial to We acknowledge the funding support received by improved clinical outcome. Current treatments offer SMaRT from Leukaemia and Lymphoma Research. effective means to target bulk disease, but they are Specialist Programme New Grant Award 12053: unable to eradicate residual disease, resulting in Novel Targets and Therapeutic Combinations in eventual disease relapse in the majority of patients. Myeloma. Understanding the genetics related to this residual disease population may allow more targeted treat- fl ment approaches aimed at total eradication. Con ict of Interest statement Current genetic testing strategies employed for The authors have no potential conflicts of interest. MM, or MM trials, across the UK are ad hoc and dependent upon regional commissioning arrange- References ments. There is a requirement for the production of 1. Swerdlow SH. WHO Classification of Tumours of best practice guidelines for the genetic analysis of Haematopoietic and Lymphoid Tissues, 4th edn. Lyon: patients with myeloma. Initially, it is likely that International Agency for Research on Cancer, 2008. genetic testing will be restricted to those patients with 2. Kyle RA, Rajkumar SV. Multiple myeloma. N Engl J confirmed plasma cell myeloma, by the presence of Med 2004;351:1860–73. 28 P. J. Talley et al., 2015, Vol. 113

3. Oyajobi BO. Multiple myeloma/hypercalcemia. Arthritis 21. Sawyer JR. The prognostic significance of cytogenetics Res Ther 2007;9(Suppl. 1):S4. and molecular profiling in multiple myeloma. Cancer 4. Palumbo A, Anderson K. Multiple myeloma. N Engl Genet 2011;204:3–12. J Med 2011;364:1046–60. 22. Uchiyama H, Barut BA, Mohrbacher AF, et al. Adhesion 5. http://www.cancerresearchuk.org/cancer-info/cancerstats/ of human myeloma-derived cell lines to bone marrow types/myeloma/uk-multiple-myeloma-statistics (5 January stromal cells stimulates interleukin-6 secretion. Blood 2015, date last accessed). 1993;82:3712–20. 6. Kyle RA, Elveback LR. Management and prognosis of 23. Fonseca R, Bergsagel PL, Drach J, et al. International multiple myeloma. Mayo Clin Proc 1976;51:751–60. Myeloma Working Group molecular classification of

7. Kronke J, Udeshi ND, Narla A, et al. Lenalidomide multiple myeloma: spotlight review. Leukemia Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 causes selective degradation of IKZF1 and IKZF3 in mul- 2009;23:2210–21. tiple myeloma cells. Science 2014;343:301–5. 24. Bergsagel PL, Chesi MV. Molecular classification and 8. Lu G, Middleton RE, Sun H, et al. The myeloma drug risk stratification of myeloma. Hematol Oncol 2013;31 lenalidomide promotes the cereblon-dependent destruc- (Suppl. 1):38–41. tion of Ikaros proteins. Science 2014;343:305–9. 25. Smadja NV. Hypodiploidy is a major prognostic factor 9. Tagoug I, Jordheim LP, Herveau S, et al. Therapeutic in multiple myeloma. Blood 2001;98:2229–38. enhancement of ER stress by insulin-like growth factor I 26. Quesnel B. Multiple myeloma: all roads lead to cyclin D. sensitizes myeloma cells to proteasomal inhibitors. Clin Blood 2005;106:1–2. Cancer Res 2013;19:3556–66. 27. Bergsagel PL, Kuehl WM, Zhan F, et al. Cyclin D dysre- 10. Vincenz L, Jager R, O’Dwyer M, et al. Endoplasmic gulation: an early and unifying pathogenic event in mul- reticulum stress and the unfolded protein response: tar- tiple myeloma. Blood 2005;106:296–303. geting the Achilles heel of multiple myeloma. Mol 28. Chesi M, Nardini E, Brents LA, et al. Frequent transloca- Cancer Ther 2013;12:831–43. tion t(4;14)(p16.3;q32.3) in multiple myeloma is asso- 11. White-Gilbertson S, Hua Y, Liu B. The role of endoplas- ciated with increased expression and activating mic reticulum stress in maintaining and targeting mul- mutations of fibroblast growth factor receptor 3. Nat tiple myeloma: a double-edged sword of adaptation and Genet 1997;16:260–4. apoptosis. Front Genet 2013;4:109. 29. Chesi M, Nardini E, Lim RS, et al. The t(4;14) transloca- 12. Fletcher CDM. Who Classification of Tumours of Soft tion in myeloma dysregulates both FGFR3 and a novel Tissue and Bone, 4th edn. Lyon: International Agency gene, MMSET, resulting in IgH/MMSET hybrid tran- for Research on Cancer, 2013. scripts. Blood 1998;92:3025–34. 13. Bird JM, Owen RG, D’Sa S, et al. Guidelines for the diag- 30. Lopez-Corral L, Gutierrez NC, Vidriales MB, et al. The nosis and management of multiple myeloma 2011. Br J progression from MGUS to smoldering myeloma and Haematol 2011;154:32–75. eventually to multiple myeloma involves a clonal expan- 14. Kuppers R, Klein U, Hansmann ML, et al. Cellular sion of genetically abnormal plasma cells. Clin Cancer origin of human B-cell lymphomas. N Engl J Med Res 2011;17:1692–700. 1999;341:1520–9. 31. Leone PE, Walker BA, Jenner MW, et al. Deletions of 15. LeBien TW, Tedder TF. B lymphocytes: how they CDKN2C in multiple myeloma: biological and clinical develop and function. Blood 2008;112:1570–80. implications. Clin Cancer Res 2008;14:6033–41. 16. Pasqualucci L, Neumeister P, Goossens T, et al. Hyper- 32. Ross FM, Avet-Loiseau H, Ameye G, et al. Report from mutation of multiple proto-oncogenes in B-cell diffuse the European Myeloma Network on interphase FISH in large-cell lymphomas. Nature 2001;412:341–6. multiple myeloma and related disorders. Haematologica 17. Kuehl WM, Bergsagel PL. Multiple myeloma: evolving 2012;97:1272–7. genetic events and host interactions. Nat Rev Cancer 33. An G, Xu Y, Shi L, et al. t(11;14) multiple myeloma: a 2002;2:175–87. subtype associated with distinct immunological features, 18. Fairfax KA, Kallies A, Nutt SL, et al. Plasma cell develop- immunophenotypic characteristics but divergent outcome. ment: from B-cell subsets to long-term survival niches. Leuk Res 2013;37:1251–7. Semin Immunol 2008;20:49–58. 34. Ross FM, Chiecchio L, Dagrada G, et al. The t(14;20) is 19. Morgan GJ, Walker BA, Davies FE. The genetic architecture a poor prognostic factor in myeloma but is associated of multiple myeloma. Nat Rev Cancer 2012;12:335–48. with long-term stable disease in monoclonal gammopa- 20. Shapiro-Shelef M, Calame K. Regulation of plasma-cell thies of undetermined significance. Haematologica development. Nat Rev Immunol 2005;5:230–42. 2010;95:1221–5. Review of genetic screening in myeloma, 2015, Vol. 113 29

35. Walker BA, Leone PE, Chiecchio L, et al. A compendium 46. Walker BA, Leone PE, Jenner MW, et al. Integration of of myeloma-associated chromosomal copy number global SNP-based mapping and expression arrays reveals abnormalities and their prognostic value. Blood key regions, mechanisms, and genes important in the 2010;116:e56–65. pathogenesis of multiple myeloma. Blood 2006;108: 36. Avet-Loiseau H, Li C, Magrangeas F, et al. Prognostic 1733–43. signiﬁcance of copy-number alterations in multiple 47. Chapman MA, Lawrence MS, Keats JJ, et al. Initial myeloma. J Clin Oncol 2009;27:4585–90. genome sequencing and analysis of multiple myeloma. 37. Walker BA, Wardell CP, Brioli A, et al. Translocations Nature 2011;471:467–72. at 8q24 juxtapose MYC with genes that harbor super- 48. Keats JJ, Fonseca R, Chesi M, et al. Promiscuous muta-

enhancers resulting in overexpression and poor pro- tions activate the noncanonical NF-kappaB pathway in Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 gnosis in myeloma patients. Blood Cancer J 2014;4: multiple myeloma. Cancer Cell 2007;12:131–44. e191. 49. Heath DJ, Chantry AD, Buckle CH, et al. Inhibi- 38. Cremer FW, Bila J, Buck I, et al. Delineation of distinct ting Dickkopf-1 (Dkk1) removes suppression of bone for- subgroups of multiple myeloma and a model for clonal mation and prevents the development of osteolytic bone evolution based on interphase cytogenetics. Genes disease in multiple myeloma. J Bone Miner Res 2009; Chromosomes Cancer 2005;44:194–203. 24:425–36. 39. Tricot G, Barlogie B, Jagannath S, et al. Poor prognosis 50. Bolli N, Avet-Loiseau H, Wedge DC, et al. Heterogeneity in multiple myeloma is associated only with partial or of genomic evolution and mutational profiles in multiple complete deletions of chromosome 13 or abnormalities myeloma. Nat Commun 2014;5:2997. involving 11q and not with other karyotype abnormal- 51. Dimopoulos K, Gimsing P, Gronbaek K. Aberrant micro- ities. Blood 1995;86:4250–6. RNA expression in multiple myeloma. Eur J Haematol 40. Avet-Loiseau H, Daviet A, Saunier S, et al. Chro- 2013;91:95–105. mosome 13 abnormalities in multiple myeloma are 52. Broderick P, Chubb D, Johnson DC, et al. Common vari- mostly monosomy 13. BrJHaematol2000;111: ation at 3p22.1 and 7p15.3 influences multiple myeloma 1116–7. risk. Nat Genet 2012;44:58–61. 41. Gutierrez NC, Castellanos MV, Martin ML, et al. Prog- 53. Koura DT, Langston AA. Inherited predisposition to nostic and biological implications of genetic abnormal- multiple myeloma. Ther Adv Hematol 2013;4: ities in multiple myeloma undergoing autologous stem 291–7. cell transplantation: t(4;14) is the most relevant adverse 54. Chubb D, Weinhold N, Broderick P, et al. Common prognostic factor, whereas RB deletion as a unique variation at 3q26.2, 6p21.33, 17p11.2 and 22q13.1 abnormality is not associated with adverse prognosis. influences multiple myeloma risk. Nat Genet 2013; Leukemia 2007;21:143–50. 45:1221–5. 42. Drach J, Ackermann J, Fritz E, et al. Presence of a p53 55. Gerlinger M, Rowan AJ, Horswell S, et al. Intra- gene deletion in patients with multiple myeloma predicts tumor heterogeneity and branched evolution revealed by for short survival after conventional-dose chemotherapy. multiregion sequencing. N Engl J Med 2012;366: Blood 1998;92:802–9. 883–92. 43.ChangH,NingY,QiX,etal.Chromosome1p21 56. Pratt G, Jenner M, Owen R, et al. Updates to the guide- deletion is a novel prognostic marker in patients lines for the diagnosis and management of multiple with multiple myeloma. Br J Haematol 2007;139: myeloma. Br J Haematol 2014;167:131–3. 51–4. 57. Chng WJ, Van Wier SA, Ahmann GJ, et al. A validated 44. Boyd KD, Ross FM, Walker BA, et al. Mapping of FISH trisomy index demonstrates the hyperdiploid and chromosome 1p deletions in myeloma identifies FAM46C nonhyperdiploid dichotomy in MGUS. Blood 2005; at 1p12 and CDKN2C at 1p32.3 as being genes in regions 106:2156–61. associated with adverse survival. Clin Cancer Res 2011; 58. Tonon G, Anderson KC. Disentangling the myeloma 17:7776–84. web. Clin Cancer Res 2011;17:7210–2. 45. Wu KL, Beverloo B, Lokhorst HM, et al. Abnormalities 59. Alpar D, de Jong D, Holczer-Nagy Z, et al. Multiplex of chromosome 1p/q are highly associated with chromo- ligation-dependent probe amplification and fluores- some 13/13q deletions and are an adverse prognostic cence in situ hybridization are complementary tech- factor for the outcome of high-dose chemotherapy in niques to detect cytogenetic abnormalities in multiple patients with multiple myeloma. Br J Haematol 2007; myeloma. Genes Chromosomes Cancer 2013;52: 136:615–23. 785–93. 30 P. J. Talley et al., 2015, Vol. 113

60. Shendure J, Ji H. Next-generation DNA sequencing. Nat 64. Braggio E, Fonseca R. Unraveling the multiple myeloma Biotechnol 2008;26:1135–45. genome in the next-generation sequencing era: challenges 61. Raffan E, Semple RK. Next generation sequencing– to translating knowledge into the clinic. Expert Rev implications for clinical practice. Br Med Bull 2011;99: Hematol 2011;4:579–81. 53–71. 65. Egan JB, Shi CX, Tembe W, et al. Whole-genome 62. Quail MA, Smith M, Coupland P, et al. A tale of three sequencing of multiple myeloma from diagnosis to next generation sequencing platforms: comparison of Ion plasma cell leukemia reveals genomic initiating Torrent, Paciﬁc Biosciences and Illumina MiSeq sequen- events, evolution, and clonal tides. Blood 2012;120: cers. BMC Genomics 2012;13:341. 1060–6.

63. Lohn Z, Adam S, Birch PH, et al. Incidental ﬁndings 66. Luo J, Solimini NL, Elledge SJ. Principles of cancer Downloaded from https://academic.oup.com/bmb/article/113/1/15/284455 by guest on 27 September 2021 from clinical genome-wide sequencing: a review. J Genet therapy: oncogene and non-oncogene addiction. Cell Couns 2014;23:463–473. 2009;136:823–37.