MOLECULAR BIOLOGY


 

The cell of origin is a post-germinal center cell, which expresses immunoglobulin heavy chain (IgH) postswitch isotypes. The post-follicular origin of myeloma cells is indicated by the fact that the immunoglobulin gene sequences are somatically hypermutated. Most myeloma clones have been selected for by an antigen.

Multiple myeloma is due to an illegitimate class switch recombination in the non-functioning allele of the immunoglobulin heavy chain (IgH) locus. Translocations at the IgH locus (14q32) are the most common karyotypic abnormalities in MM. Since the transcription of the IgH locus is very active in B cells and plasma cells, transfer of a potential oncogene to 14q32 through the translocation, will result in the dysregulation of the oncogene. A switch recombination is legitimate during isotype switching, when cells need to produce a functional Ig allele. In MM, the rearrangements are due to an illegitimate switch recombination. A legitimate switch recombination is an essential prerequisite for generating antibody diversity and producing a functional repertoire to prevent infections, Therefore, in evolutionary terms, the development of myeloma is the price to pay for the production of a functional immune system.

The IgH genes contain 44 variable (V), 27 diversity (D), and 6 joining (J) gene segments. In the IgH rearrangement, D joins J, then V joins DJ. The rearrangement of the Ig heavy chain gene (IgH) precedes the rearrangement of the Ig light chain gene (IgL). The CDR3, used in the detection of minimal residual disease by PCR, consists of V-N-D-N-J, where N are the nucleotides inserted by the terminal deoxynucleotidyltransferase during the VDJ rearrangement in the B-cell differentiation. The sequence and length of N varies in each clone. 

In MM cells, there is no ongoing somatic hypermutation, but they can have new mutations throughout the VDJ-S region.

Myeloma is a malignancy characterized by complex genetic aberrations. The genomic evolution of myeloma cells is heterogeneous, because most cases have  a complex subclonal structure, with linear evolution, branching evolution, and differential subclonal response to therapy.

 

 

TRANSLOCATIONS IN MM

 t(14;20)  (q32;q12)  MAFB

<1%

 t(4;14)  (p16;q32)  MMSET/FGFR3

 15%

 t(6;14)  (p21;q32)  Cyclin D3

2%

 t(8;14)  (q24;q32)  MAFA

<1%

 t(11;14)  (q13;q32)  Cyclin D1

15%

 t(12;14)  (p13;q32)  Cyclin D2

<1%

 t(14;16)  (q32;q23)  c-MAF

5%

 

Most cases of MM show translocations involving the Ig heavy-chain (IgH) gene (chrom. 14q32) and:
 -Bcl-1 (cyclin D1) (chrom. 11)
 -Fibroblastic growth factor receptor 3 (FGFR3) (chrom. 4)
 -c-maf oncogene (chrom. 16)

In many cases, these translocations are karyotypically silent.

The translocations result in the juxtaposition of an oncogene to the IgH enhancers, with consequent overexpression of the oncogene.

 

TRANSLOCATIONS
See specific section - CYTOGENETICS AND FISH.

 

The major growth factor for myeloma cells is IL-6. It is secreted by BM stromal cells (major source) and myeloma cells.
IL-6 transgenic mice develop  plasmacytomas in lymph nodes, Peyer's patches, and spleen.

Dendritic cells cultured from myeloma patients contain HHV-8. This virus encodes an IL-6 homologue. Several studies did not confirm these findings, and therefore there is no evidence that HHV-8 plays a role in the pathogenesis of MM.

 

 

Isotype class switching and the pathogenesis of multiple myeloma.
Hematol Oncol. 2002 Jun;20(2):75-85.
Fenton JA, Pratt G, Rawstron AC, Morgan GJ.
[Review]

Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma.
Blood. 2005 Jul 1;106(1):296-303.
Bergsagel PL, Kuehl WM, Zhan F, Sawyer J, Barlogie B, Shaughnessy J Jr.
Authors propose that cyclin D dysregulation is an unifying pathogenic event in MM. MM translocations dysregulate:
 - CCND1 (11q13). Biallelic dysregulation of CCND1 occurs in nearly 40% of tumors, most of which hyperdiploid.
 - CCND2, targeted by the transcription factors MAF (16q23) and MAFB (20q11). CCND2 expression can be increased either with or without a t(4;14) translocation.
 - CCND3 (6p21)

Molecular pathogenesis and a consequent classification of multiple myeloma.
J Clin Oncol. 2005 Sep 10;23(26):6333-8.
Bergsagel PL, Kuehl WM.
[Review]

The molecular classification of multiple myeloma.
Blood. 2006 Sep 15;108(6):2020-8.
Zhan F, Huang Y, Colla S, Stewart JP, Hanamura I, Gupta S, Epstein J, Yaccoby S, Sawyer J, Burington B, Anaissie E, Hollmig K, Pineda-Roman M, Tricot G, van Rhee F, Walker R, Zangari M, Crowley J, Barlogie B, Shaughnessy JD Jr.

Authors performed mRNA expression profiles in plasma cells from 414 newly diagnosed MM patients. They validated 7 disease subtypes, characterized by known genetic lesions: c-MAF- and MAFB-, CCND1- and CCND3-, MMSET-activating translocations, and hyperdiploidy.

Immunoglobulin gene rearrangements and the pathogenesis of multiple myeloma.
Blood. 2007 Nov 1;110(9):3112-21.
González D, van der Burg M, García-Sanz R, Fenton JA, Langerak AW, González M, van Dongen JJ, San Miguel JF, Morgan GJ.
[Review]

Analysis of clonotypic switch junctions reveals multiple myeloma originates from a single class switch event with ongoing mutation in the isotype-switched progeny.
Blood. 2008 Sep 1;112(5):1894-903.
Taylor BJ, Kriangkum J, Pittman JA, Mant MJ, Reiman T, Belch AR, Pilarski LM.
Authors found that myeloma cells have a single, unchanging clonotypic switch junction that persists over time. They suggest that post-switch plasma cells arise from a single clonogenic IgM cell, and that MM-PC progenitors reside in the post-switch population. They also found that aggressive clones that evolve throughout the course of MM are associated with new mutations upstream of the switch junction, often targeting the intronic enhancer, a key control region in IgH expression.

Initial genome sequencing and analysis of multiple myeloma.
Nature. 2011 Mar 24;471(7339):467-72.
Chapman MA, Lawrence MS, Keats JJ, Cibulskis K, Sougnez C, Schinzel AC, Harview CL, Brunet JP, Ahmann GJ, Adli M, Anderson KC, Ardlie KG, Auclair D, Baker A, Bergsagel PL, Bernstein BE, Drier Y, Fonseca R, Gabriel SB, Hofmeister CC, Jagannath S, Jakubowiak AJ, Krishnan A, Levy J, Liefeld T, Lonial S, Mahan S, Mfuko B, Monti S, Perkins LM, Onofrio R, Pugh TJ, Rajkumar SV, Ramos AH, Siegel DS, Sivachenko A, Stewart AK, Trudel S, Vij R, Voet D, Winckler W, Zimmerman T, Carpten J, Trent J, Hahn WC, Garraway LA, Meyerson M, Lander ES, Getz G, Golub TR.

Characterization of IGH locus breakpoints in multiple myeloma indicates a subset of translocations appear to occur in pregerminal center B cells.
Blood. 2013 Apr 25;121(17):3413-9.
Walker BA, Wardell CP, Johnson DC, Kaiser MF, Begum DB, Dahir NB, Ross FM, Davies FE, Gonzalez D, Morgan GJ.
In this study of translocation analysis in 61 myeloma samples, the authors found that 100% of the t(4;14) translocations were mediated by class switch recombination in mature B cells in the germinal center. Unexpectedly, 21% of the t(11;14) and 25% of the t(14;20) were generated during the DH-JH recombination in pro-B-cells in the bone marrow.

Heterogeneity of genomic evolution and mutational profiles in multiple myeloma.
Nat Commun. 2014 Jan 16;5:2997.
Bolli N1, Avet-Loiseau H2, Wedge DC3, Van Loo P4, Alexandrov LB3, Martincorena I3, Dawson KJ3, Iorio F5, Nik-Zainal S6, Bignell GR3, Hinton JW3, Li Y3, Tubio JM3, McLaren S3, O' Meara S3, Butler AP3, Teague JW3, Mudie L3, Anderson E3, Rashid N7, Tai YT7, Shammas MA8, Sperling AS7, Fulciniti M7, Richardson PG7, Parmigiani G9, Magrangeas F10, Minvielle S10, Moreau P11, Attal M12, Facon T13, Futreal PA14, Anderson KC7, Campbell PJ1, Munshi NC8.

Next-generation sequencing of peripheral B-lineage cells pinpoints the circulating clonotypic cell pool in multiple myeloma.
Blood. 2014 Jun 5;123(23):3618-21.
Thiele B, Kloster M, Alawi M, Indenbirken D, Trepel M, Grundhoff A, BinderM.
Until this study, some expert speculated that the clonogenic population of malignant plasma cells consisted of less differentiated pre-switch B cells. Instead, these authors demonstrated that the clonogenic cells are differentiated post-switch plasma cells (which circulate in the peripheral blood). 

APOBEC family mutational signatures are associated with poor prognosis translocations in multiple myeloma.
Nat Commun. 2015 Apr 23;6:6997.
Walker BA, Wardell CP, Murison A, Boyle EM, Begum DB, Dahir NM, Proszek PZ, Melchor L, Pawlyn C, Kaiser MF, Johnson DC, Qiang YW, Jones JR, Cairns DA, Gregory WM, Owen RG, Cook G, Drayson MT, Jackson GH, Davies FE, Morgan GJ.
The authors did whole exomic sequence of 463 cases of newly diagnosed multiple myeloma. They found an APOBEC mutational signature in 4% of cases. They were associated to the translocations t(14;16) and t(14;20), which result in deregulation of the MAF and MAFB genes, respectively. APOBEC is a family of enzymes editing the DNA, and they deaminate cytosine to uracil in single-stranded DNA.
  - In MAF and MAFB, the mutations were only seen in the basic-leucine zipper domain
  - In FGFR3, the mutations were scattered throughout the gene

Single-molecule analysis reveals widespread structural variation in multiple myeloma.
Proc Natl Acad Sci U S A. 2015 Jun 23;112(25):7689-94.
Gupta A, Place M, Goldstein S, Sarkar D, Zhou S, Potamousis K, Kim J, Flanagan C, Li Y, Newton MA, Callander NS, Hematti P, Bresnick EH, Ma J, Asimakopoulos F, Schwartz DC.

Clonal selection and double-hit events involving tumor suppressor genes underlie relapse in myeloma.
Blood. 2016 Sep 29;128(13):1735-44.
Weinhold N, Ashby C, Rasche L, Chavan SS, Stein C, Stephens OW, Tytarenko R, Bauer MA, Meissner T, Deshpande S, Patel PH, Buzder T, Molnar G, Peterson EA, van Rhee F, Zangari M, Thanendrarajan S, Schinke C, Tian E, Epstein J, Barlogie B, Davies FE, Heuck CJ, Walker BA, Morgan GJ.

Circulating tumour DNA analysis demonstrates spatial mutational heterogeneity that coincides with disease relapse in myeloma.
Leukemia. 2017 Aug;31(8):1695-1705.
Mithraprabhu S, Khong T, Ramachandran M, Chow A, Klarica D, Mai L, Walsh S, Broemeling D, Marziali A, Wiggin M, Hocking J, Kalff A, Durie B, Spencer A.
This is an interesting study, which compares mutational status of malignant plasma cells in the bone marrow aspirate (the traditional analysis) with the one in the circulating free tumor DNA. Paired bone marrow and peripheral blood were analyzed in 48 patients. The results favor the peripheral blood analysis, not only because it was a noninvasive test, but also because it revealed a higher frequency of mutations. Evidently, myeloma consists of a heterogeneous population of malignant cells which may have different clonal evolution both in time and space, even due to the heterogeneous and multifocal involvement of the bone marrow. A bone marrow aspirate from a single focus may fail to reveal the genomic complexity of the whole neoplasm.

Spatial genomic heterogeneity in multiple myeloma revealed by multi-region sequencing.
Nat Commun. 2017 Aug 16;8(1):268.
Rasche L, Chavan SS, Stephens OW, Patel PH, Tytarenko R, Ashby C, Bauer M, Stein C, Deshpande S, Wardell C, Buzder T, Molnar G, Zangari M, van Rhee F, Thanendrarajan S, Schinke C, Epstein J, Davies FE, Walker BA, Meissner T, Barlogie B, Morgan GJ, Weinhold N.
This study shows results of sequencing in both bone marrow and distant focal lesions of 51 patients with multiple myeloma. They demonstrated that there is a high degree of genomic heterogeneity between myeloma clones, as "spatial" differences were found in more than 75% of patients. The results supported the notion that patients with advanced disease have a more significant genomic heterogeneity between different anatomical sites.

 

 


 

BRAF

Although BRAF mutation is a rare molecular event in patients with multiple myeloma (about 3%), it is more frequent in patients with extramedullary disease.

BRAF V600E mutation in early-stage multiple myeloma: good response to broad acting drugs and no relation to prognosis.
Blood Cancer J. 2015 Mar 20;5:e299.
Rustad EH, Dai HY, Hov H, Coward E, Beisvag V, Myklebost O, Hovig E, Nakken S, Vodák D, Meza-Zepeda LA, Sandvik AK, Wader KF, Misund K, Sundan A, Aarset H, Waage A.
In this study, the BRAF V600E mutation was detected in 11 (5%) of 209 patients with multiple myeloma. Its presence was not associated with an adverse prognosis.

Vemurafenib in Multiple Nonmelanoma Cancers with BRAF V600 Mutations.
N Engl J Med. 2015 Aug 20;373(8):726-36.
Hyman DM, Puzanov I, Subbiah V, Faris JE, Chau I, Blay JY, Wolf J, Raje NS, Diamond EL, Hollebecque A, Gervais R, Elez-Fernandez ME, Italiano A, Hofheinz RD, Hidalgo M, Chan E, Schuler M, Lasserre SF, Makrutzki M, Sirzen F, Veronese ML, Tabernero J, Baselga J.
In this study, 122 patients with cancers positive for the BRAF V600 mutation received treatment with vemurafenib. Five of them had myeloma - no responses were observed.

 

CXCR4/SDF-1

Mechanisms of regulation of CXCR4/SDF-1 (CXCL12)-dependent migration and homing in multiple myeloma.
Blood. 2007 Apr 1;109(7):2708-17.
Alsayed Y, Ngo H, Runnels J, Leleu X, Singha UK, Pitsillides CM, Spencer JA, Kimlinger T, Ghobrial JM, Jia X, Lu G, Timm M, Kumar A, Côté D, Veilleux I, Hedin KE, Roodman GD, Witzig TE, Kung AL, Hideshima T, Anderson KC, Lin CP, Ghobrial IM.
This study found that CXCR4 is expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. The CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. AMD3100 inhibited the homing of MM cells to the bone marrow niches. Authors  demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing.

 

HGF

The hepatocyte growth factor/Met pathway controls proliferation and apoptosis in multiple myeloma.
Leukemia. 2003 Apr;17(4):764-74.
Derksen PW, de Gorter DJ, Meijer HP, Bende RJ, van Dijk M, Lokhorst HM, Bloem AC, Spaargaren M, Pals ST.
The receptor for the hepatocyte growth factor/scatter factor (HGF) is Met, which is expressed on MM cells. HGF is produced by BM stromal cells and by some MM cell lines, enabling para- or autocrine interaction. Authors found that Met is expressed by the majority of MM cell lines and by approximately half of the primary plasma cell neoplasms tested. HGF is a potent myeloma growth and survival factor, by activating the RAS/mitogen-activated protein kinase and PI3K/PKB pathways. HGF had anti-apoptotic effects on both MM cell lines and primary MM cells.

 

IGF

Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications.
Oncogene. 2002 Aug 22;21(37):5673-83.
Mitsiades CS, Mitsiades N, Poulaki V, Schlossman R, Akiyama M, Chauhan D, Hideshima T, Treon SP, Munshi NC, Richardson PG, Anderson KC.
Authors demonstrate that insulin-like growth factor-1 (IGF-1) stimulates sustained activation of NF-kappaB and Akt. In contrast, IL-6 did not cause sustained NF-kappaB activation, and it induced less pronounced Akt activation.

Insulin-like growth factor I induces migration and invasion of human multiple myeloma cells.
Blood. 2004 Jan 1;103(1):301-8.
Qiang YW, Yao L, Tosato G, Rudikoff S.
Insulin-like growth factor I (IGF-I) is one of several growth factors that promote the growth of MM cells. This study assesses the ability of IGF-I to serve additionally as a chemotactic factor affecting the mobility and invasion of MM cells. Authors demonstrate that IGF-I promotes transmigration through vascular endothelial cells and bone marrow stromal cell lines.

Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade.
Mol Cancer Ther. 2005 Oct;4(10):1533-40.
Shi Y, Yan H, Frost P, Gera J, Lichtenstein A.

The role of IGF-1 as a major growth factor for myeloma cell lines and the prognostic relevance of the expression of its receptor.
Blood. 2009 May 7;113(19):4614-26.
Sprynski AC, Hose D, Caillot L, Réme T, Shaughnessy JD Jr, Barlogie B, Seckinger A, Moreaux J, Hundemer M, Jourdan M, Meissner T, Jauch A, Mahtouk K, Kassambara A, Bertsch U, Rossi JF, Goldschmidt H, Klein B.
These authors investigated 5 myeloma growth factors (IL-6, IGF-1, HGF, HB-EGF, and APRIL) in serum-free cultures of MM cell lines. IGF-1 was the major one.

 

IL-6

The interleukin-6 receptor alpha-chain (CD126) is expressed by neoplastic but not normal plasma cells.
Blood. 2000 Dec 1;96(12):3880-6.
Rawstron AC, Fenton JA, Ashcroft J, English A, Jones RA, Richards SJ, Pratt G, Owen R, Davies FE, Child JA, Jack AS, Morgan G.
Interleukin-6 (IL-6) induces proliferation and inhibits apoptosis of neoplastic plasma cells. Authors determined the IL-6R expression levels on plasma cells from 93 patients with MM, 66 patients with MGUS or plasmacytoma, and normal plasma cells in 11 cases. CD126, i.e., the IL-6R alpha chain, was not expressed in normal plasma cells, but it was expressed in the neoplastic plasma cells in about 90% of MM patients. In patients with MGUS or plasmacytoma, neoplastic plasma cells expressed higher levels of CD126 when compared with the normal plasma cells from the same patients.

IL-6 transgenic mouse model for extraosseous plasmacytoma.
Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1509-14.
Kovalchuk AL, Kim JS, Park SS, Coleman AE, Ward JM, Morse HC 3rd, Kishimoto T, Potter M, Janz S.
IL-6 is plays an important role in growth, differentiation, and survival of B cells. These authors tested the contribution of IL-6 to the development of extramedullary (extraosseous) plasmacytomas (PCT) by generating BALB/c mice carrying an IL-6 transgene. All mice developed plasmacytosis and lymphoproliferation. By 18 months of age, over half developed readily transplantable PCT in lymph nodes, Peyer's patches, and occasionally spleen. These neoplasms had a t(12;15) translocation. Surprisingly, about 30% of the mice developed germinal center-derived NHL (follicular and diffuse large cell B cell lymphomas), that often coexisted with PCT.

 

MET

Phase II study of the c-MET inhibitor tivantinib (ARQ 197) in patients with relapsed or relapsed/refractory multiple myeloma.
Ann Hematol. 2017 Jun;96(6):977-985.
Baljevic M, Zaman S, Baladandayuthapani V, Lin YH, de Partovi CM, Berkova Z, Amini B, Thomas SK, Shah JJ, Weber DM, Fu M, Cleeland CS, Wang XS, Stellrecht CM, Davis RE, Gandhi V, Orlowski RZ.
This is a phase II study of tivantinib, a c-MET inhibitor, in 16 patients with relapsed/refractory myeloma. There was no response, but 4 of 11 evaluable patients (36%) had stable disease.

 

MYC

Heterogeneous pattern of chromosomal breakpoints involving the MYC locus in multiple myeloma.
Genes Chromosomes Cancer. 2003 Jul;37(3):261-9.
Fabris S, Storlazzi CT, Baldini L, Nobili L, Lombardi L, Maiolo AT, Rocchi M, Neri A.
MM and plasma cell leukemia (PCL) can harbor c
hromosomal rearrangements of MYC (8q24), which may involve the Ig loci. Author used dual-color FISH to characterize the breakpoint locations of chromosomal rearrangements of the MYC locus in 14 MM cell lines, 66 cases of MM and 4 cases of PCL. They found MYC locus alterations in 21 cases: MYC/Ig (mainly IgH) fusions in 11 cell lines, 2 MM patients, and 1 PCL patient, and extra signals and/or abnormal MYC localizations in 5 MM patients and 2 PCL patients. These data document the dispersion of 8q24 breakpoints in MM.

IL-6 and MYC collaborate in plasma cell tumor formation in mice.
Blood. 2010 Mar 4;115(9):1746-54.
Rutsch S, Neppalli VT, Shin DM, DuBois W, Morse HC 3rd, Goldschmidt H, Janz S.

Chromosome 8q24.1/c-MYC abnormality: a marker for high-risk myeloma.
Leuk Lymphoma. 2014 Aug 18:1-6.
Glitza IC1, Lu G, Shah R, Bashir Q, Shah N, Champlin RE, Shah J, Orlowski RZ, Qazilbash MH.
This study of 23 patients with myeloma and c-MYC rearrangement showed aggressive clinical features of this subset of myeloma: 12 patients had plasma cell leukemia (primary or secondary) or extrameduallary disease, and the median overall survival of the entire group was only 20 months.

Chromosome 8q24.1/c-MYC abnormality: a marker for high-risk myeloma.
Leuk Lymphoma. 2015 Mar;56(3):602-7.
Glitza IC, Lu G, Shah R, Bashir Q, Shah N, Champlin RE, Shah J, Orlowski RZ, Qazilbash MH.
The oncogene c-MYC is rearranged in about 15% of patients with multiple myeloma. In this study, 23 patients with myeloma and c-MYC rearrangement had an aggressive clinical course, because of the high incidence of plasma cell leukemia and/or extramedullary disease either at diagnosis or upon progression (12 patients, 52%), and a short median survival (only 20 months).

 

 


Giampaolo Talamo, M.D.