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Reports 2020; volume 12:9054

The molecular pathogenesis small pre-clinical proliferation will be of multiple myeloma observed.2 Further acquisition of additional Corresponding Authors: Claudio Cerchione, variants may then dictate evolution from Hematology Unit, Istituto Scientifico these small clonal proliferations, never rec- Romagnolo per lo Studio e la Cura dei Tumori 1,2 3 Niccolò Bolli, Giovanni Martinelli, ognized in clinical practice, to a clinically (IRST) IRCCS, Via Piero Maroncelli 40, 3 Claudio Cerchione evident following natural selection 47014, Meldola (FC), Italy. 1 acting on the resulting phenotypic Tel. +39.0543.739100 Department of and Hemato- E-mail: [email protected] diversity.3 The complex multicellular Oncology, University of Milan, Italy; Niccolò Bolli, Hematology Unit, Fondazione 2 Hematology Unit, Fondazione IRCCS microenvironment, the competition for IRCCS Ca’ Granda Ospedale Maggiore Ca’ Granda Ospedale Maggiore metabolites, oxygen, growth factors, and Policlinico, Milan, Italy; Department of the necessity for immune escape4 will also Policlinico, Milan, Italy; 3Hematology Oncology and Hemato-Oncology, University dictate which is the fittest for growth. Unit, Istituto Scientifico Romagnolo per of Milan, Via Francesco Sforza, 35 Milan, Genomic plasticity, conferred by the loss of Italy. lo Studio e la Cura dei Tumori (IRST) DNA-repair mechanisms and/or acquisition Tel. +390255033337 IRCCS, Meldola, Italy of a hypermutator phenotype, will certainly E-mail: [email protected] facilitate the ability to adapt of the tumor cells. Key words: Multiple myeloma, tumor evolu- dyscrasias are frequent tion, next-generation sequencing, personal- Abstract ized medicine. hematological malignancies, and are usual- Multiple Myeloma (MM) is character- ly regarded to as a more complex disease Received for publication: 14 December 2020. ized by uncontrolled proliferation and accu- from a genomic point of view as compared Accepted for publication: 15 December 2020. mulation of clonal plasma cells within the to and .5 The most marrow. However, the cell of origin is frequent conditions, forming a continuous Conflict of interest: the Authors declare no a B-lymphocyte acquiring aberrant genomic spectrum that can often be observed over potential conflict of interest. events in the of a lymph time in the same patient, are clinically cate- node as off-target events during somatic- gorized as of This work is licensed under a Creative hypermutation and class-switch recombina- unknown significance (MGUS), smoldering Commons Attribution-NonCommercial 4.0 tion driven by activation-induced-deami- multiple myeloma (SMM) and active MM. International License (CC BY-NC 4.0). nase. Whether pre-germinal center events The diagnosis of MGUS requires the ©Copyright: the Author(s), 2020 are also required for transformation, and presence of a serum monoclonal of Licensee PAGEPress, Italy which additional events are required for dis- <3 g/dL and <10% clonal bone marrow Hematology Reports 2020; 12:9054 ease progression is still matter of debate. As (BM) plasma cells, in the absence of myelo- doi:10.4081/hr.2020.9054 early treatment in asymptomatic phases is ma defining events or . MGUS may progress to the more advanced asymp- gaining traction in the clinic, a better under- mopathies will be described in the context tomatic stage of SMM, defined by a serum standing of the molecular pathogenesis of of the normal B-cell development. We will monoclonal protein of ≥3 g/dL or 24h uri- myeloma progression would allow stratifi- then focus on the different patterns of evo- nary monoclonal protein ≥500 mg, and/or cation of patients based on their risk of pro- lution from asymptomatic to aggressive 10–60% clonal BM plasma cells in the gression, thus rationalizing efficacy and stages of disease and the impact MM het- absence of myeloma defining events or cost of clinical interventions. In this review, erogeneity has in this process. we will discuss the development of MM, amyloidosis. Active MM in turn is diag- from the cell of origin through asympto- nosed in presence of clonal BM plasma matic stages such as monoclonal gammopa- cells >10% and/or a proven bony or extra- medullary , and one or thy of undetermined significance and smol- Initiating events dering MM, to the development of sympto- more myeloma-defining events: end-organ matic disease. We will explain the genetic damage (hypercalcemia, renal failure, ane- Asymptomatic clonal expansion of heterogeneity of MM, one of the major mia, lytic bone ), ≥60% bone mar- hematopoietic cells are nowadays well rec- drivers of disease recurrence. In this con- row clonal plasma cells, serum free light- ognized at the expense of plasma cells,7 text, moreover, we will propose how this chain (FLC) ratio ≥100 (for kappa) or <0.01 having been found decades ago through the 6 knowledge may influence future diagnostic (for lambda), and >1 focal on MRI. identification of a monoclonal in and therapeutic interventions. These categories reflect differences in the serum through protein . management to prevent the development of On the contrary, the presence of an asymp- end-organ damage, or its prompt recogni- tomatic clonal B-lymphocytosis has been 6 Introduction tion and treatment. From a genomic point identified more recently thanks to flow of view, the question then is whether this cytometry.8 Stem cells can also show simi- Random mutagenesis is a frequent and clinical evolution is paralleled by a similar lar instances of asymptomatic clonal expan- likely ubiquitous phenomenon in replicat- biological evolution of the neoplastic clone, sions, identified through next-generation ing tissues, stemming from slight intrinsic from initiating lesions to those associated sequencing (NGS).9,10 All these evidences infidelity of DNA replication and repair with progression and development of an confirm the multi-step nature of cancer evo- processes, and enzymatic modification of aggressive disease, and whether this can be lution. In fact, it is thought that all MMs are DNA bases.1 Additionally, exogenous exploited clinically. preceded by an MGUS stage, even if not processes may increase this rate. In this review, we will describe recent clinically evident.11 Rarely, these events will result in creation advances on the molecular pathogenesis of Contrary to lymphoproliferative dis- of a variant conferring a proliferative or sur- MM. Cell-intrinsic factors involved in the eases, are not classi- vival advantage to the cell. In such case, a initiation processes of monoclonal gam- fied based on the cell of origin, since the lat-

[Hematology Reports 2020; 12:9054] [page 55] Review ter – a post-germinal center B-lymphocyte- is thought to derive from a single abnormal towards transformation conferred by kary- is morphologically different from the neo- cell cycle duplication.19 However, analysis otypic events that can be assessed by FISH plastic cell encountered in the of the activity of mutational processes with or SNP-arrays.25–27 Furthermore, the fact at the time of diagnosis – a plasma cell. a constant mutation rate on trisomic chro- that many CNAs can be found at the sub- Events leading to transformation of a mosomes showed that the number of pre- clonal level confirms their acquisition after naïve B cells upon antigen encounter within gain and post-gain if often differ- the PC clone has been established. the germinal center (GC) of lymph nodes ent from chromosome to chromosome. This NGS has the potential to allow a much are thought to arise from errors during implies that different can be deeper analysis of the genome of MGUS, class-switch-recombination (CSR) and acquired in different time windows.20 highlighting initiating events beyond recur- somatic hypermutation (SHM) of the B-cell Furthermore, only mutations in HD chro- rent translocations and CNAs. However, receptor (BCR). These are two processes mosomes acquired before the gain show an single-cell RNAseq studies have clearly aimed at increasing antigen affinity to a off-target AID signature, while mutations highlighted how at this stage there is a large peculiar antigen and conferring specific acquired after the gain don’t show any sign number of contaminating, non-clonal PC effector functions, catalyzed by the activa- of AID activity.21 This demonstrates a ger- which may hamper bulk cell analysis.28 tion-induced-deaminase (AID) enzyme. minal center origin of the . Last, However, initial targeted DNA-sequencing Through the creation of double strand mechanisms linking trisomy to neoplastic studies highlighted recurrent mutations in breaks and mutations, AID activity is at risk transformation are unclear, but may be the myeloma genes NRAS, BRAF, KRAS, of off-target mutations and rearrangements.12 linked to the expression of with- DIS3, EGR1 and LTB. Mutations were less It is unknown if the transformed cell would in the duplicated chromosomes.22 frequent than in active MM and within each need some priming in the form of pre-exist- The same analysis of the activity of case allelic frequencies were suggestive of a ing mutations or genomic lesions permis- mutational signatures with a constant activ- late acquisition of the mutation.27,29 sive to the survival upon this AID off-target ity over time has provided bases to enquire Importantly, no mutations have been detect- activity, but this is subject of intense when the transformation happens in the life ed in tumor suppressor genes such as TP53, research. Another question is whether this of the patient. Using serial samples from the or in genes involved in DNA repair mecha- transformation is favored by a germline pre- same patients, the activity of these muta- nisms as ATM or ATR usually enriched in disposition. Indeed, the risk of developing tional processes could be extrapolated back more advanced phases of the disease. a plasma cell dyscrasia is increased two- in time, concluding that the transforming Indeed, MGUS does not display an early fold in relatives of MM patients,13 and event could take already in the second or and specific single-nucleotide mutational germline transmission of several risk alleles third decade of life.21 Subsequently, activity that may explain expansion of the has been described.14,15 decades of clonal proliferation and acquisi- tumor clone. This is very different, for The transformed B-cell will then home tion of additional events would ensue example, from what observed in NPM1- to the BM and differentiate into a plasma before the clone becomes clinically evident mutated acute myeloid ,30–32 and is cell, giving rise to the clonal expansion clin- in the form of MGUS. more in line with a slowly evolving disease ically recognized as MGUS. Importantly, driven by structural events as seems to be crucial to this process is the interaction with the case for most mature lymphoid neo- the microenviroment.16 plasms. MGUS therefore displays common Genomic features of MGUS Very recently, through the combination genetic features with MM: it carries either of multi-parametric flow-sorting strategy recurrent translocations of oncogenes with In MGUS, differently from MM, clonal and low-input whole genome library prepa- switch regions of the IGH locus or an BM plasma cells are low to absent, the ration, the genome of highly purified clonal hyperdiploid (HD) karyotype. The latter monoclonal protein in the serum is low, and MGUS PCs has been sequenced.33 With the consists in multiple trisomies of odd chro- there are no signs of end-organ damage, addition of SMM and MM cases, a compre- mosomes with the exception of chromo- active MM or amyloidosis.23 hensive analysis of progressive vs non-pro- somes 13 and 17.17 IGH translocations are IGH translocations and HD are trans- gressive asymptomatic cases and their com- caused by aberrant CSR promoted by AID, forming events, however they are not suffi- parison to active MM cases has been possi- as proved by the fact that the rearrangement cient for MM development. In fact, MGUS ble. Results suggested a quite striking dif- hotspot is close to the canonical CSR break- may display these abnormalities and remain ference between MGUS and SMM cases points. Furthermore, translocation partner clinically stable. This is the case for the that remained stable in the long term. Stable genes show mutations with a signature of majority of cases, since MGUS progresses asymptomatic conditions displayed very lit- AID-induced mutations, consistent with a at an average rate of 1% of cases each year, tle activity of mutational processes besides germinal center origin of the event.18 and this rate does not increase even after the AID-activity responsible for disease ini- Subsequent to the translocation, Ig decades.24 This argues against a model of tiation,34 along with reduced numbers of enhancers promote the overexpression of continuous acquisition of additional lesions CNAs. Interesting, while the number of tri- the recurrent partner genes, consisting in the to drive progression, and on the contrary somies in HD cases were not significantly known oncogenes CCND1, WHSC1, MAF, suggests that clonal sweeps may be driven different between the various conditions, MAFB, CCND3 in t(11:14), t(4:14), by stochastic events. The question is there- stable asymptomatic cases showed fewer t(14:16), t(14:20) and t(6;14) respectively. fore what additional genomic events are instances of chr(1q) gain or amplifications, Being initiating events, IGH translocations required for progression. From a point of del(6q), gain(8q24) involving the are almost always clonal, mutually exclu- view of prevalence, the t(11;14) is more fre- locus, del(16q) as compared to progressive sive with each other and with the HD karyo- quent in MGUS, while all other transloca- cases. Finally, structural variants and partic- type.12,19 On the contrary, mechanisms lead- tions are more prevalent in MM. On the ularly complex events like chromothripsis ing to the generation of an HD karyo- contrary, del(13) and other copy-number and templated insertions20 were strikingly type are less clear. In hyperdiploid acute alterations (CNAs) are more prevalent in enriched in progressive cases, suggesting lymphoblastic leukemia, the HD karyotype MM. This suggests a differential propensity that in the future a molecular signature may

[page 56] [Hematology Reports 2020; 12:9054] Review prognosticate indolent asymptomatic cases came from the whole-genome analysis of additional genomic lesions. much better than the current clinical and paired samples from ultra-high risk SMM More recent studies on much larger laboratory parameters. progressing to MM. At baseline, the genom- sample cohorts have further expanded these ic structure of SMM was similar to MM in findings, and translated them into informa- terms of driver events. This included tion that could be used in clinical practice. translocations, CNAs and gene mutations In particular, MYC translocations41 or MYC Genomic features of SMM but particularly complex structural events. abnormalities, mutations in MAPK genes or Known secondary CNAs such as del(13q), DNA repair genes and the t(4;14) all inde- SMM carries a higher disease burden 42 del(6q), del(8p), del(16q) and amp(1q) were pendently predicted progression to MM. than MGUS, as by definition clonal plasma also frequent. Differently from MGUS, the This evidence makes it tempting to assume cells in the BM must be >10% and <60%. structure of high-risk SMM was therefore that genomics can really help prognostica- The rate of progression of SMM is 10% per very similar to that of MM.39 Comparing tion of SMM by identifying at diagnosis year in the first 5 years, then declines to 3% the genome of paired samples, clonal evolu- cases that will behave like MGUS, and will for the next 5 and to 1% after ten year from tion followed one of two main modalities. rarely progress in years owing to the acqui- diagnosis.35,36 SMM is therefore quite het- Authors described a “static progression sition of additional genomic events, and erogeneous from a clinical point of view, model”, where the subclonal structure cases that are de facto MM and already suggesting its definition includes patients grows unchanged from SMM to MM, and a show all features of an aggressive neo- ranging from an actual active MM that does plasms. This also highlights the inadequacy not yet satisfy criteria for diagnosis to oth- “spontaneous evolution” model, where the subclonal structure of SMM changes of current prognostic scores, mostly based ers with a biologically indolent form similar on the tumor burden of SMM43. to MGUS just with more BM PCs. because of the acquisition of one or more MGUS patients do not routinely under- subclones and/or loss of others at the time go BM examinations during follow-up, of progression to MM. On average, patients would progress in less than one year in the therefore it is unusual to catch an evolution Genomic features of MM from MGUS to SMM even if this is what it static model, and at a much slower pace in is supposed to happen in all progressing the spontaneous evolution model. Genomic studies in MM have much cases. This makes it hard to ascertain events Furthermore, analysis of mutational changed the perception of the disease in the associated with initial progression of this processes active in each subclone was also last 10 years. Dozens of mutated genes, asymptomatic conditions, and most of what particularly revealing. AID activity was pre- mutational processes, CNAs and complex we know about SMM comes from cases ponderant in the ancestral clone of each structural events have been added to the diagnosed ab initio as such. Furthermore, case, again confirming a germinal center genomic landscape of what initially seemed since SMM itself can be stable for years, origin of the disease. Subclones evolved to be a disease with few karyotypic events44 our knowledge of its evolution is biased later in the disease course and responsible and gene mutations. Initial enthusiasm for towards more aggressive and more rapidly for progression showed instead enriched the discovery of actionable mutations such evolving cases. activity of the APOBEC family of DNA as BRAF V600E45 has nevertheless been From a genomic point of view, SMM deaminases, an aberrant mutational process curbed by the evidence that MM at diagno- appears to carry similar genetic abnormali- active across a variety of cancers40, shed- sis is a highly heterogeneous disease,18,46,47 ties to active MM, just at a lower ding some light onto aberrant genomic so that targeted treatment can trigger rapid frequency.29,37,38 An interesting observation processes responsible for the acquisition of subclonal outgrowth outcompeting the main

Table 1. Main genomic features of MGUS (1a), SMM (1b) and MM (1c). Stage of disease Genomic features Stable asymptomatic cases Fewer instances of chr(1q) gain or amplifications, del(6q), gain(8q24) involving the MYC locus, del(16q) as compared to progressive cases Progressive cases Structural variants and particularly complex events like chromothripsis and templated insertions are strikingly enriched Table 1a MGUS

Genomic feature Clinical significance MYC abnormalities/translocations, Indipendently predict SMM progression to MM MAPK or DNA repair genes mutations t (4; 14) Table 1b SMM

Genomic feature Clinical significance Mutations in CRBN t (11;14) Predict IMIDs and PI resistance Predict targeted treatment () responsiveness High-risk lesions: Simultaneously resistance to PIs and IMiDs and worse prognosis - bi-allelic events in tumor suppressors, - amp(1q), - acquisition of an APOBEC signature Table 1c MM

[Hematology Reports 2020; 12:9054] [page 57] Review clone at diagnosis.48 At the same time, this with response to a specific treatment or lack advantage in cases with t(11;14) but a dis- wealth of information has failed to improve thereof. However, no such biomarkers have advantage in other subgroups73 will open a biological classification of the disease. been found to correlate with response to the field for personalized treatment in MM This is still based on the main cytogenetic inhibitors (PIs) or immunomod- based on patient-specific gene lesions. events, confirming these are the initiating ulatory drugs (IMiDs), the two most used Furthermore, several trials are exploring a events that shape the subsequent trajectory drug classes in induction. Indeed, mutations risk-adapted approach, if not a “basket” of evolution, providing some constraint on in CRBN in IMiD-resistant cases59 and in design where personalized treatment is the type of alterations required for progres- proteasome subunit genes in PI-resistant offered based on each patient’s gene muta- sion20. HD and IGH-translocated cases still cases60 account for a tiny fraction of cases tions.74 The most immediate application of provide the mainstay of classification. and are not found -or are found at the sub- NGS to the clinic will nevertheless be that Within HD cases, about a third associate clonal level- in the majority of cases that do of reliable measurement of minimal-resid- with CNAs – mostly del(1p), amp(1q), not respond to such treatments.50,61 Indeed, ual disease through the sequencing of the del(13q), del(14q), del(16q) – and the other analysis of cases that are simultaneously patient-specific B-cell receptor rearrange- two thirds with mutations, with a prepon- resistant to PIs and IMiDs suggested instead ment. The prognostic value of this tech- derance of mutations of the RAS family. that chemoresistance in MM is achieved nique seems to be extremely high,75 and this T(4;14) cases cluster in two categories, fre- through the acquisition of high-risk lesions, is likely explained by the fact that this tech- quently associate with del(13q), and in the such as bi-allelic events in tumor suppres- nique may overcome the heterogeneity of first they also associate with CNAs, in the sors, amp(1q), and acquisition of an other phenotypic and genotypic markers of second they show fewer CNAs but muta- APOBEC signature.50 The described sub- the tumor clone. tions in DIS3 and FGFR3. t(11:14) cases clonal heterogeneity is responsible for this Limits of NGS in MM could be repre- also fall in two different categories, one dynamic evolution of the tumor through sented by the difficulty of obtaining enough with CCND1 and IRF4 mutation, and one lines of treatment and is especially visible DNA from bone marrow aspirates, and by 18,62 with TP53 bi-allelic inactivation. A seventh in cases of extra-medullary evolution. the spatial heterogeneity of the disease.62 In category is not characterized by any partic- this respect, analysis of circulating tumor ular structural event, but by a hypermutated cells or cell-free tumor DNA could repre- genotype.20 This evidence reinforces the sent a suitable alternative for longitudinal notion that gene mutations are late events, Potential clinical applications of disease monitoring. While initial approach- whose impact is not strong enough to define genomic technologies in plasma es in MGUS and SMM have been in part 76,77 a genetic category of the disease. cell dyscrasias disappointing, MM at diagnosis seem to Furthermore, most mutated genes are not offer more circulating cells and cfDNA thus even expressed in MM.49,50 On the contrary, The recent progress prompted by allowing a more informative analysis.78,79 and similar to solid , structural genomics discoveries in MM raised the Limiting amounts of circulating DNA seem events – many of which are complex and question as to whether these merit incorpo- also to limit the analysis of peripheral blood non-recurrent, yet impacting recurrent driv- ration into routine clinical practice. The par- to track disease response to treatment so er genes – are the events that drive and adigm has been set by myeloid malignan- far80. However, there is little doubt that 51 define the disease. cies and particularly AML, where genomics knowledge banks built on thousands of However, genomic analysis has has dramatically impacted classification63 cases of MM, including genomic and clini- revealed several important prognostic cor- and prognostication,64 prompting the devel- cal details are highlighting prognostic relates, most of which are not captured by opment of clinical-grade NGS sequencing groups that can’t be captured by FISH 52 the R-ISS . A well-known example is what panels.65–67 In MM, the nature of the alone.55,53 More such efforts are underway is somehow improperly referred to as “dou- genome of the disease requires that translo- and will likely soon reach a consensus on a ble-hit” multiple myeloma, i.e. cases with cation in IGH regions and copy-number reduced set of genomic lesions that may ISS stage 3 and chr(1q)amp, or cases with abnormalities are captured along with gene explain most of the risk of MM at diagnosis 53 TP53 bi-allelic inactivation. The defini- mutations, posing additional hurdles to the and may amenable to routine clinical-grade tion is somewhat improper since it has been design of the panel. While several NGS detection. Furthermore, the advent of new observed how several combinations of attempts have been successful at matching drug classes will improve the treatment genomic events have prognostic relevance, or outperforming the accuracy of FISH for landscape of MM, but potential benefits 68–70 some showing a clear interaction, others the detection of such structural events, may be offset by increased costs and toxic- 54,55 simply highlighting an additive effect. the perception still is that NGS is a much ity. This mandates that novel biomarkers are Other events associated with worse progno- complicated technique and the extra-infor- found to rationalize treatment, implying that sis include a high activity of the APOBEC mation added to common FISH panels is genomic analysis will become routine clini- 56 mutational process, IGL-MYC transloca- not going to change the way we make clin- cal practice at diagnosis and at each 57 58 tions, TP53 mutations. While this list is ical decision soon. relapse.81 not at all inclusive, the main examples are This perception may soon be chal- cited to stress the point that the use of lenged. Starting from SMM, novel prognos- genomic analysis for MM prognostication tic markers incorporating cytogenetic 71 is still in its infancy, and a comprehensive events have been validated, accepting the References analysis will be required over large datasets notion that disease biology should be more to understand the independent prognostic relevant than disease burden for SMM 1. Alexandrov LB, Nik-Zainal S, Wedge role of these and other variables to inform prognostication.72 In newly diagnosed MM, DC, et al. Signatures of mutational pro- clinical decisions. treatment paradigms still follow a “one-size cesses in human cancer. 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