CYTOGENETICS AND FISH

 

Metaphase cytogenetics in multiple myeloma is informative in only 25-30% of patients. The technique is limited by:
 - The low degree of proliferation of malignant plasma cells (usually 1% or less are cycling)
 - The frequently limited degree of bone marrow infiltration, and the fact that myeloma is a patchy disease
   Therefore, the analyzed metaphases often belong to normal hematopoietic cells
 - Presence of cryptic translocations
Instead, fluorescence in situ hybridization (FISH) of interphase nuclei can detect chromosomal abnormalities in most cases (about 80%). However, FISH is limited by the need for a pre-established selection of target genes, and it does not provide a complete analysis of the genetic abnormalities.

 

Fluorescence in situ hybridization analysis of aneuploidization patterns in monoclonal gammopathy of undetermined significance versus multiple myeloma and plasma cell leukemia.
Cancer. 2003 Feb 1;97(3):601-9.
Rasillo A, Tabernero MD, Sánchez ML, Pérez de Andrés M, Martín Ayuso M, Hernández J, Moro MJ, Fernández-Calvo J, Sayagués JM, Bortoluci A, San Miguel JF, Orfao A.
This study assessed by interphase fluorescence in situ hybridization (FISH) the incidence of numerical abnormalities of chromosomes 6, 9, 13, and 17 in 30 patients with MGUS, and it compared the results with those found in 158 MM patients and 20 PCL patients. 57% of the MGUS patients displayed abnormalities for at least 1 of the 4 chromosomes analyzed, while 75% of MM and PCL cases had the same abnormalities. Compared with MM patients, MGUS patients had a lower incidence of +9 (23% vs. 54%) and 13q deletions (21% vs. 38%.

A pooled analysis of karyotypic patterns, breakpoints and imbalances in 783 cytogenetically abnormal multiple myelomas reveals frequently involved chromosome segments as well as significant age- and sex-related differences.
Br J Haematol. 2003 Mar;120(6):960-9.
Nilsson T, Höglund M, Lenhoff S, Rylander L, Turesson I, Westin J, Mitelman F, Johansson B.
This study analyses the cytogenetic features of 783 abnormal multiple myeloma cases. Hyperdiploidy was most common, followed by hypodiploidy, pseudodiploidy and tri-/tetraploidy. Most cases had a complex karyotype, with a median of 8 abnormalities per patient. There were no sex- or age-related differences regarding the number of abnormalities. The most frequent genomic breakpoints were: 14q32, 11q13, 1q10, 8q24, 1p11, 1q21, 22q11, 1p13, 1q11, 19q13, 1p22, 6q21 and 17p11. The most frequent imbalances were +9, -13, +15, +19, +11, and -Y. -Y as the sole change was more common in elderly men.

Cytogenetics of multiple myeloma: interpretation of fluorescence in situ hybridization results.
Br J Haematol. 2003 Mar;120(6):944-52.
Harrison CJ, Mazzullo H, Cheung KL, Gerrard G, Jalali GR, Mehta A, Osier DG, Orchard KH.
These authors studied cytogenetics and/or interphase FISH on bone marrow samples or purified plasma cells from 37 MM patients. Abnormal karyotypes by multiplex FISH were found in 11 patients, all of which were highly complex. Interphase FISH showed:
 - Translocations involving the IgH locus in 16 (43%) patients
 - The translocation t(11;14)(q13;q32), resulting in the IgH/cyclin D1 (CCND1) gene fusion, was seen in 12 (32%) of patients
 - Other rearrangements of IgH were seen in 4 (11%) patients
 - 14 patients had additional copies of chromosome 11
 - 20 patients (54%) had 13q14 deletions, 10 of whom also had t(11;14) or another IgH translocation.

Frequent gain of chromosome band 1q21 in plasma-cell dyscrasias detected by fluorescence in situ hybridization: incidence increases from MGUS to relapsed myeloma and is related to prognosis and disease progression following tandem stem-cell transplantation.
Blood. 2006 Sep 1;108(5):1724-32.
Hanamura I, Stewart JP, Huang Y, Zhan F, Santra M, Sawyer JR, Hollmig K, Zangari M, Pineda-Roman M, van Rhee F, Cavallo F, Burington B, Crowley J, Tricot G, Barlogie B, Shaughnessy JD Jr.
These authors used FISH to investigate the amplification of chromosome band 1q21 (Amp1q21) in more than 500 untreated patients with plasma cell dyscrasias. They found that Amp1q21 is associated with both disease progression and poor prognosis. The frequency of Amp1q21 was 0% in MGUS, 45% in smoldering MM, 43% in newly diagnosed MM, and 72% in relapsed MM. Amp1q21 was an independent poor prognostic factor at multivariate analysis. Patients with newly diagnosed MM with Amp1q21 had a 5-year EFS of 38% and OS of 52%, while patients lacking Amp1q21 had a 5-year EFS of 62% and OS of 78%. 5-year post-relapse survival was 15% in patients with relapsed MM who had Amp1q21, and 53% in patients lacking Amp1q21 at relapse. At relapse, both proportion of cells with Amp1q21 and copy number of 1q21 were higher than those at diagnosis.

Interphase fluorescence in situ hybridization on selected plasma cells is superior in the detection of cytogenetic aberrations in plasma cell dyscrasia.
Genes Chromosomes Cancer. 2010 Nov;49(11):991-7.
Put N, Lemmens H, Wlodarska I, Konings P, Moreau Y, Hagemeijer A, Vandenberghe P, Michaux L.

 

 


t(11;14) TRANSLOCATION

 

The t(11;14) translocation is the most common translocation detected in MM. The t(11;14) occurs not only in some cases of MM, but also in most cases of mantle cell lymphoma. In both cases, the IgH locus is involved, and cyclin D1 is overexpressed. However, the molecular mechanism is different: while the t(11;14) translocation in mantle cell lymphoma is caused by errors in the VDJ-mediated recombination (the breakpoint is located into or near a JH segment), the t(11;14) in MM is caused by errors in the switch-mediated recombination (the breakpoint is located in the IgH switch regions). Therefore, myeloma cells have already undergone lgH switch recombination, whereas mantle-cell lymphoma cells have not. The t(11;14) translocation is the hallmark of IgM, IgE, and nonsecretory MM (but not IgD MM).
The t(11;14) is associated with:
  - Hyposecretory disease
  - Lymphoplasmacytic morphology
  - CD20 expression
  - IgM MM (>90%)

 

Myeloma and the t(11;14)(q13;q32); evidence for a biologically defined unique subset of patients.
Blood. 2002 May 15;99(10):3735-41.
Fonseca R, Blood EA, Oken MM, Kyle RA, Dewald GW, Bailey RJ, Van Wier SA, Henderson KJ, Hoyer JD, Harrington D, Kay NE, Van Ness B, Greipp PR.
These authors determine the clinical and biologic significance of the t(11;14) translocation using FISH in 336 MM patients. 53 (16%) patients were positive for t(11;14)(q13;q32). These patients were:
 - were more likely to have a serum M protein of <1 g/dL (28% vs 15%)
 - had a lower plasma cell labeling index
 - were less likely to be hyperdiploid by DNA content analysis (14% vs 62%)
 - had a better survival and response to treatment (but this did not reach statistical significance)

Translocation t(11;14)(q13;q32) is the hallmark of IgM, IgE, and nonsecretory multiple myeloma variants.
Blood. 2003 Feb 15;101(4):1570-1.
Avet-Loiseau H, Garand R, Lodé L, Harousseau JL, Bataille R; Intergroupe Francophone du Myélome.
These authors analyzed a series of 8 IgM, 9 IgD, 2 IgE, and 14 nonsecretory (NS) MM cases using FISH. A very high incidence (83%) of t(11;14) was detected in:
 - IgM MM cases (7 of 8)
 - IgE MM cases (2 of 2)
 - NS MM cases (11 of 14)
 - Not in IgD MM cases (2 of 9)
t(11;14) was associated with 2 features: "lymphoplasmacytic" presentation mainly in IgM MM and a lower secreting capacity in the others.
These authors showed that t(11;14) is the hallmark of IgM, IgE, and NS MM, (but not IgD MM).

Translocation t(11;14) in multiple myeloma: Analysis of translocation breakpoints on der(11) and der(14) chromosomes suggests complex molecular mechanisms of recombination.
Genes Chromosomes Cancer. 2004 Feb;39(2):151-5.
Fenton JA, Pratt G, Rothwell DG, Rawstron AC, Morgan GJ.
These authors describe the characterization of the genomic DNA breakpoints of 2 MM patients with the t(11;14). They show that the IgH translocations are more complex than simple reciprocal events mediated by errors in class-switch recombination. DNA from chromosome band 11q13 joined to already recombined hybrid switch region sequences, and, in 1 case, a further rearrangement occurred at the t(11;14) recombination site.

The enigmatic role of cyclin D1 in multiple myeloma.
Int J Cancer. 2005 Jun 10;115(2):171-6.
Lesage D, Troussard X, Sola B.
[Review]

The CCND1 c.870G>A polymorphism is a risk factor for t(11;14)(q13;q32) multiple myeloma.
Nat Genet. 2013 Apr 26;45(5):522-5.
Weinhold N, Johnson DC, Chubb D, Chen B, Försti A, Hosking FJ, Broderick P, Ma YP, Dobbins SE, Hose D, Walker BA, Davies FE, Kaiser MF, Li NL, Gregory WA, Jackson GH, Witzens-Harig M, Neben K, Hoffmann P, Nöthen MM, Mühleisen TW, Eisele L, Ross FM, Jauch A, Goldschmidt H, Houlston RS, Morgan GJ, Hemminki K.
This study indicates that genetic factors confer a risk of a specific chromosomal translocation.

 

 


t(4;14) TRANSLOCATION

 

The t(4;14) (p16;q32) chromosomal translocation is the second most common translocation in myeloma, and it is associated with the worst prognosis. It results in the formation of a fusion IgH-MMSET (multiple myeloma SET domain) transcript in the der(4) chromosome.
SET = Suppressor of variegation, Enhancer of zeste, and Trithorax.
This translocation may induce overexpression of the fibroblast growth factor receptor 3 (FGFR3) gene. All cases overexpress MMSET, but up to 25% of cases do not overexpress FGFR3. FGFR3 mutations can occur even in the absence of the t(4;14). These mutations produce a constitutively active receptor, which shows dimerization and autophosphorylation even in the absence of ligand. The deletion of MMSET is associated with the malformation syndrome called Wolf-Hirschhorn syndrome. MMSET is a histone methyltransferase (methylation of histones allows cells to undergo DNA damage repair). It expression has been shown to promote cellular adhesion and clonogenic growth in multiple myeloma. MMSET-high cells repair DNA damage at an enhanced rate and have a higher proliferation rate as compared to MMSET-low cells.
In normal conditions, FGFR3 inhibits chondrocyte growth. Achondroplasia, the common form of dwarfism, is caused by genetic mutations that activate the FGFR3 receptor tyrosine kinase.

 

A molecular study of the t(4;14) in multiple myeloma.
Br J Haematol. 2002 Aug;118(2):514-20.
Sibley K, Fenton JA, Dring AM, Ashcroft AJ, Rawstron AC, Morgan GJ.
These authors analysed 67 MM cases and 13 MGUS cases, using RT-PCR to detect IgH-MMSET fusions. They found the t(4;14) translocation in 7 of 67 (10%) MM cases, and all 7 expressed FGFR3, a phenomenon not seen in t(4;14)-negative MM cases. MGUS cases had a similar proportion of t(4;14) (2 of 13; 15%), but none of these expressed FGFR3. No mutations of the FGFR3 gene were found in the the MM or MGUS samples, except for a single case of relapsed MM, which was negative for mutations 13 months earlier, but it developed a K650E mutation in the kinase domain of FGFR3 at relapse.

Novel mutation and RNA splice variant of fibroblast growth factor receptor 3 in multiple myeloma patients at diagnosis.
Haematologica. 2002 Oct;87(10):1036-40.
Soverini S, Terragna C, Testoni N, Ruggeri D, Tosi P, Zamagni E, Cellini C, Cavo M, Baccarani M, Tura S, Martinelli G.
These authors investigated the presence of FGFR3 expression and activating mutations in a series of 78 newly diagnosed MM patients, using RT-PCR and gene sequence. RT-PCR revealed FGFR3 mRNA expression in 10 of 78 (13%) patients. In 2 cases, sequence analysis revealed FGFR3 mutations. Interestingly, a patient without the t(4;14) had 3 additional, abnormal-sized transcripts, corresponding to truncated transcripts originating from alternative splicing.

Genomic characterization of the chromosomal breakpoints of t(4;14) of multiple myeloma suggests more than one possible aetiological mechanism.
Oncogene. 2003 Feb 20;22(7):1103-13.
Fenton JA, Pratt G, Rawstron AC, Sibley K, Rothwell D, Yates Z, Dring A, Richards SJ, Ashcroft AJ, Davies FE, Owen RG, Child JA, Morgan GJ.
These authors characterized the genomic breakpoints of t(4;14) translocations from 7 MM patients. In 5 patients, chromosome 14q32 breakpoints were located in the IgH mu switch (S) region with deletion of intervening DNA from aberrant class switch recombination (CSR). However, in 2 patients the rearranged hybrid switch region sequence was joined to DNA from chromosome 4p16. Therefore, IgH translocations can occur in B cells that have already undergone legitimate CSR. These findings suggest that primary IgH translocations involve different mechanisms, and they may occur at different time points in the development of malignant plasma cells, either during the physiological CSR or at a later stage.

A subset of multiple myeloma harboring the t(4;14)(p16;q32) translocation lacks FGFR3 expression but maintains an IGH/MMSET fusion transcript.
Blood. 2003 Mar 15;101(6):2374-6.
Santra M, Zhan F, Tian E, Barlogie B, Shaughnessy J Jr.

The reciprocal t(4;14)(p16;q32) translocation activates expression of FGFR3 and creates an IgH/MMSET fusion transcript. These authors analyzed 32 (18%) of 178 patients with newly diagnosed MM harboring the t(4;14)(p16;q32). They found that 32% of these cases lacked expression of FGFR3, but they still expressed MMSET and had the IgH/MMSET fusion transcript. Therefore, the oncogenic event of this recurrent translocation in MM is the activation of MMSET, not FGFR3.

CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the potential treatment of t(4;14) multiple myeloma.
Blood. 2005 Apr 1;105(7):2941-8.
Trudel S, Li ZH, Wei E, Wiesmann M, Chang H, Chen C, Reece D, Heise C, Stewart AK.
This study describes the therapeutic efficacy of CHIR-258, an inhibitor of FGFR3, in a xenograft mouse model of FGFR3+ MM.

Overexpression of transcripts originating from the MMSET locus characterizes all t(4;14)(p16;q32)-positive multiple myeloma patients.
Blood. 2005 May 15;105(10):4060-9.
Keats JJ, Maxwell CA, Taylor BJ, Hendzel MJ, Chesi M, Bergsagel PL, Larratt LM, Mant MJ, Reiman T, Belch AR, Pilarski LM.
Only transcripts originating from the WHSC1/MMSET/NSD2 gene are uniformly dysregulated in MM patients with the t(4;14) translocation. The transcripts detected were:
 - Multiple myeloma SET domain containing protein (MMSET I)
 - MMSET II
 - Exon 4a/MMSET III
 - Response element II binding protein (RE-IIBP)
These are produced by alternative transcription initiation events and alternative splicing.
The Exon 4a/MMSET III splice variant contains a protein domain that prevents nucleolar localization.
RE-IIBP is universally dysregulated and also potentially functional in all t(4;14) patients, regardless of FGFR3 expression or breakpoint type.

The multiple myeloma associated MMSET gene contributes to cellular adhesion, clonogenic growth, and tumorigenicity.
Blood. 2008 Jan 15;111(2):856-64.
Lauring J, Abukhdeir AM, Konishi H, Garay JP, Gustin JP, Wang Q, Arceci RJ, Matsui W, Park BH.
These authors provided evidence that MMSET plays a significant role in t(4;14) MM cells.
 - Down-regulation of MMSET expression in MM cell lines reduced colony formation in methylcellulose
 - The effect in liquid culture was modest
 - MMSET knockdown led to cell-cycle arrest of adherent MM cells and reduced the ability of MM cells to adhere to extracellular matrix.
 - MMSET knockdown and knockout reduced tumor formation by MM xenografts

The MMSET protein is a histone methyltransferase with characteristics of a transcriptional corepressor.
Blood. 2008 Mar 15;111(6):3145-54.
Marango J, Shimoyama M, Nishio H, Meyer JA, Min DJ, Sirulnik A, Martinez-Martinez Y, Chesi M, Bergsagel PL, Zhou MM, Waxman S, Leibovitch BA, Walsh MJ, Licht JD.
 -
MMSET possesses domains found within chromatin regulators, including the SET domain
 - MMSET possesses methyltransferase activity for core histone H3 lysine 4 and histone 4 lysine 20
 - MMSET-mediated repression was associated with increased H4K20 methylation gene and loss of histone acetylation
 - MMSET formed a complex with HDAC1 and HDAC2, mSin3a, and the histone demethylase LSD1
 - MMSET coexpression enhanced HDAC1- and HDAC2-mediated repression
 - Knockdown of MMSET compromised viability of a myeloma cell line
This study indicate that MMSET influences gene expression as a direct modifier of chromatin and through binding with other chromatin-modifying enzymes.

MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells.
Haematologica. 2009 Jan;94(1):78-86.
Brito JL, Walker B, Jenner M, Dickens NJ, Brown NJ, Ross FM, Avramidou A, Irving JA, Gonzalez D, Davies FE, Morgan GJ.
Using several biological assays, these authors found that MMSET regulates the expression of genes involved in:
 - Cell cycle progression (CCND2, CCNG1, BRCA1, AURKA and CHEK1)
 - Apoptosis (CASP1, CASP4 and FOXO3A)
 - Cell adhesion (ADAM9 and DSG2)

Clinical and biological features of t(4;14) multiple myeloma: a prospective study.
Leuk Lymphoma. 2011 Feb;52(2):238-46.
Karlin L, Soulier J, Chandesris O, Choquet S, Belhadj K, Macro M, Bouscary D, Porcher R, Ghez D, Malphettes M, Asli B, Brouet JC, Bories JC, Hermine O, Fermand JP, Arnulf B.
This is a report of 102 consecutive patients with myeloma expressing the t(4;14). Prognosis was poor, with median progression-free survival of 12 months, and median overall survival after transplantation of 31 months.

Ligand activation leads to regulated intramembrane proteolysis of fibroblast growth factor receptor 3.
Mol Biol Cell. 2011 Oct;22(20):3861-73.
Degnin CR, Laederich MB, Horton WA.

Phase 2 study of dovitinib in patients with relapsed or refractory multiple myeloma with or without t(4;14) translocation.
Eur J Haematol. 2015 Oct;95(4):316-24.
Scheid C, Reece D, Beksac M, Spencer A, Callander N, Sonneveld P, Kalimi G, Cai C, Shi M, Scott JW, Stewart AK.
In this study, 43 patients with relapsed or refractory myeloma were treated with dovitinib, a receptor tyrosine kinase inhibitor that targets FGFR3. Thirteen patients had the t(4;14) translocation. The results were disappointing, because no objective responses were observed.

MMSET/WHSC1 enhances DNA damage repair leading to an increase in resistance to chemotherapeutic agents.
Oncogene. 2016 Nov 10;35(45):5905-5915.
Shah MY, Martinez-Garcia E, Phillip JM, Chambliss AB, Popovic R, Ezponda T, Small EC, Will C, Phillip MP, Neri P, Bahlis NJ, Wirtz D, Licht JD.

 

 


Giampaolo Talamo, M.D.