Molecular Karyotyping by High Resolution Arrray CGH Uncovers New Deleted and Amplified Genomic Regions That Contain Genes of Biological Interest in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1540-1540
Author(s):  
Cristina Largo ◽  
Sara Alvarez ◽  
David Blesa ◽  
Felipe Prosper ◽  
M. Jose Calasanz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Resolution ◽  
Human Genome ◽  
Cell Lines ◽  
Copy Number ◽  
Gene Copy ◽  
Comparative Genomic ◽  
Myeloma Cells ◽  
Biological Interest ◽  
Copy Number Changes

Abstract Multiple Myeloma (MM) is a malignancy characterized by clonal expansion of plasma cells. In 50% of the cases, the neoplastic transformation begins with a chromosomal translocation that juxtaposes the IGH gene locus to an oncogene. After defining the IGH-translocations present in a panel of MM cell lines, we conducted expression-profiling analysis. Supervised analysis identified 166 genes significantly differentially expressed among the cell lines harboring MMSET/FGFR3 (4p16), MAF (16q) and CCND1 (11q13) rearrangements. Besides translocations, gene copy number changes are also frequent in MM but far less characterized than in other neoplasias. We conducted array Comparative Genomic Hybridization (arrayCGH) with the same cDNA platform that was used for the expression analysis with the double purpose of characterizing the amplified genome regions and identifying the amplified and overexpressed (A/O) genes within these regions. We focused in five chromosomes recurrently affected by gains/amplifications in primary samples and cell lines where we found 60 A/O genes. Among them, twenty-five (42%) were only overexpressed when amplified, and six, CHI3L1, ELMO1, BNIP3L, PLAG1, LOC157567, and VPS28, showed a significant association between overexpression and gain/amplification. In a second step, we are conducting a high resolution analysis of copy number changes in flow sorted CD138 myeloma cells from patients with normal karyotype, as stated by conventional cytogenetics. We are using the Human Genome CGH Microarray 44A platform from Agilent Technologies (Palo Alto, CA), which contains 44.000 60-mer oligonucleotides covering the human genome with an average resolution of 40 Kb. As expected, this genomic approach, performed on selected cells, allowed the identification of a high number of deletions and gains. We have found small regions (below 500 Kb of size) that were rearranged as follows: Copy number changes on selected CD138+ MM cells Chromosome Rearrangement Some genes of biological interest loss 1p12-p21.3 STXBP3, SORT1, HRMT1L6, PSMAS loss 6q24.1–q24.2 STXBP5, IL20RA, MYB loss 11q14.1–q22.3 MMP1, 3, 7, 8, 10, 12, 13, 20, BIRC2 & 3 loss 14q12–q21.3 FOXA, FBXO33, SNX6 loss 16p12.1 TP53TG3, ZNF267, CREBPB gain 1q21 CKS1B, AIM2, SELL, CHI3L1 gain 8q24.12–q24.23 C-MYC, PTK2

scholarly journals Whole-genome optical mapping of bone-marrow myeloma cells reveals association of extramedullary multiple myeloma with chromosome 1 abnormalities

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eva Kriegova ◽  
Regina Fillerova ◽  
Jiri Minarik ◽  
Jakub Savara ◽  
Jirina Manakova ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
High Risk ◽  
Copy Number ◽  
Optical Mapping ◽  
Chromosome 1 ◽  
Gene Copy ◽  
Whole Genome ◽  
Myeloma Cells ◽  
Genomic Architecture

AbstractExtramedullary disease (EMM) represents a rare, aggressive and mostly resistant phenotype of multiple myeloma (MM). EMM is frequently associated with high-risk cytogenetics, but their complex genomic architecture is largely unexplored. We used whole-genome optical mapping (Saphyr, Bionano Genomics) to analyse the genomic architecture of CD138+ cells isolated from bone-marrow aspirates from an unselected cohort of newly diagnosed patients with EMM (n = 4) and intramedullary MM (n = 7). Large intrachromosomal rearrangements (> 5 Mbp) within chromosome 1 were detected in all EMM samples. These rearrangements, predominantly deletions with/without inversions, encompassed hundreds of genes and led to changes in the gene copy number on large regions of chromosome 1. Compared with intramedullary MM, EMM was characterised by more deletions (size range of 500 bp–50 kbp) and fewer interchromosomal translocations, and two EMM samples had copy number loss in the 17p13 region. Widespread genomic heterogeneity and novel aberrations in the high-risk IGH/IGK/IGL, 8q24 and 13q14 regions were detected in individual patients but were not specific to EMM/MM. Our pilot study revealed an association of chromosome 1 abnormalities in bone marrow myeloma cells with extramedullary progression. Optical mapping showed the potential for refining the complex genomic architecture in MM and its phenotypes.

Comprehensive Genome-Wide Profile of Regional Gains and Losses in Multiple Myeloma Using Array-CGH: The 1q21 Amplification and Potential Role of the BCL-9 Gene in Multiple Myeloma Pathogenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 785-785 ◽  
Author(s):  
Ruben Carrasco ◽  
Giovanni Tonon ◽  
Cameron Brennan ◽  
Alexei Protopopov ◽  
Raktim Sinha ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Copy Number ◽  
Structural Changes ◽  
Array Cgh ◽  
Genetic Alterations ◽  
Comparative Genomic ◽  
Primary Tumors ◽  
Gains And Losses

Abstract Multiple Myeloma (MM) is characterized by a clonal proliferation of abnormal plasma cells in the bone marrow and is among the most frequent and lethal hematological diseases. In spite of significant effort towards the identification of the molecular events leading to this malignancy, the genetic alterations responsible for the pathogenesis of this disease remain poorly understood. Regional copy number alterations (CNAs) in cancer genomes have been among the most informative structural changes in cancer and have led to the discovery of many oncogenes and tumor supressor genes. Using array comparative genomic hybridization (array-CGH) and expression microarray technologies we have analyzed a large collection of cell lines and clinically annotated primary tumors. This high-resolution genomic analysis has identified all previously reported regional gains and losses as well as many novel highly recurrent genetic loci with potential biological and clinical relevance. In particular, we have identified an amplification at chromosome 1q21 as one of the most recurrent genetic changes in cell lines and in a subgroup of primary tumors. This chromosomal change has been previously implicated with disease progression. Analysis across several cell lines has allowed the identification of a Minimal Common Region (MCRs) of amplification at 1q21. Correlation between DNA copy number changes and expression profiling data has identified a limited set of candidate genes within this MCR that are amplified and overexpressed. Using shRNAi technology we have identified BCL-9 as a candidate gene residing at the 1q21 MCR. In vitro and in vivo functional data about the role of BL-9 will be presented. These data will provide critical understanding on the diverse pathways leading to Multiple Myeloma progression.

SNP Array Analysis Reveals Copy Number Alterations and Uniparental Disomy in Mantle Cell Lymphomas at High Resolution.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1585-1585
Author(s):  
Elena M. Hartmann ◽  
Itziar Salaverria ◽  
Silvia Bea ◽  
Andreas Zettl ◽  
Pedro Jares ◽  
...  
Keyword(s):  
High Resolution ◽  
Cell Lines ◽  
Copy Number ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Genetic Alterations ◽  
Array Analysis ◽  
Snp Array Analysis ◽  
Copy Number Changes ◽  
500K Array

Abstract Mantle Cell Lymphoma (MCL) is an aggressive B-Cell Non Hodgkin Lymphoma which is genetically characterized by the translocation t(11;14). This translocation leads to juxtaposition of the Cyclin D1 gene and the IgH locus, resulting in constitutive overexpression of Cyclin D1 and consecutive cell cycle dysregulation. Apart from this typical structural genetic alteration, several studies using conventional or array-based comparative genomic hybridization (CGH) reported a high number of secondary numerical genetic alterations contributing to MCL lymphomagenesis and influencing the clinical behavior. Increasingly, there is evidence that loss of heterozygosity (LOH) without copy number changes (e.g. caused by mitotic recombination between the chromosomal homologues, also referred to as acquired (partial) uniparental disomy (a(p)UPD), is an important alternative mechanism for tumor suppressor gene inactivation. However, this phenomenon is undetectable by CGH techniques. Single Nucleotide Polymorphism (SNP) based arrays allow - in addition to high resolution copy number (CN) analyses and SNP genotyping - in the same experiment the analysis of loss of heterozygosity (LOH) events and hereby enable the detection of copy neutral LOH. We analyzed the 3 t(11;14)-positive MCL cell lines Granta 519, HBL-2 and JVM-2 and 5 primary tumor specimens from untreated MCL patients with both the Affymetrix GeneChip®Human Mapping 100K and 500K array sets. In the 3 cell lines, we found an excellent agreement between the copy number changes obtained by SNP array analysis and previously published array CGH results. Extending published results (Nielaender et al., Leukemia 2006), we found regions of pUPD in all 3 MCL cell lines, which often affected regions reported as commonly deleted in MCL. Intriguingly, HBL-2 that is characterized by relatively few chromosomal losses, carries an increased number of large regions showing copy neutral LOH. Furthermore, we compared the results obtained by the 100K and 500K mapping array sets from 5 primary MCL tumor specimens with previously published conventional CGH data. All cases showed genetic alterations in both conventional CGH and SNP array analysis. The total number of copy number alterations detected by conventional CGH was 35, including 23 losses, 10 gains and 2 amplifications. The total number of CN alterations detected by the mapping 100K and 500K array sets was 81 (50 losses, 26 gains and 5 amplifications) and 82 (50 losses, 27 gains and 5 amplifications), respectively. We found an excellent agreement in the large CN alterations detected by conventional CGH and both SNP array platforms. Furthermore, we identified >40 mostly small CN alterations that have not been detected by conventional CGH (median size <5MB for losses and <3Mb for gains). The CN alterations detected by the 100k and the 500K array sets were highly identical. Importantly, we discovered regions of partial UPD in 4 of the 5 MCL cases (size range from around 2Mb up to a single region >40Mb). In conclusion, the results demonstrate the capability of SNP array analysis for identifying CN alterations and partial UPD at high resolution in MCL cell lines as well as in primary tumor samples.

Gene expression profiling and gene copy-number changes in malignant mesothelioma cell lines

2007 ◽  
Vol 46 (10) ◽  
pp. 895-908 ◽  
Author(s):  
Claudia Zanazzi ◽  
Remko Hersmus ◽  
Imke M. Veltman ◽  
Ad J.M. Gillis ◽  
Ellen van Drunen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Cell Lines ◽  
Malignant Mesothelioma ◽  
Expression Profiling ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Mesothelioma Cell ◽  
Copy Number Changes

Coordinate Interstitial Deletion of Retinoblastoma (RB1) and Neurobeachin (NBEA) Is a Recurring Event in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2480-2480
Author(s):  
Julie O’Neal ◽  
AnnaLynn Molitoris ◽  
Feng Gao ◽  
Anjum Hassan ◽  
Ryan Monahan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Copy Number ◽  
Plasma Cells ◽  
Interstitial Deletion ◽  
Oligonucleotide Array ◽  
Comparative Genomic ◽  
Copy Number Loss ◽  
Dna Copy Number ◽  
Chromosome 13

Abstract Monoallelic chromosome 13 deletion detected by cytogenetics predicts poor patient survival in multiple myeloma (MM), but the genes responsible have not been conclusively identified. To this end, we performed array comparative genomic hybridization (aCGH) using a novel, human chromosome 13 oligonucleotide array (Nimblegen) with 385,272 probes and median probe spacing of 60 base pairs. The dense coverage, and the use of germline DNA collected from each patient as internal controls for DNA copy number polymorphisms, enabled unprecedented map resolution of somatic DNA gains and losses on chromosome 13. Array CGH was performed on genomic DNA isolated from CD138+ bone marrow plasma cells purified from 20 patients with MM, monoclonal gammopathy of undetermined significance (MGUS), or amyloidosis. Visual analysis of the aCGH data identified 4 patients with chromosome 13 interstitial deletions that were confirmed using a circular binary segment algorithm (Nimblegen). Monosomy chromosome 13 was detected in 5 patients by cytogenetics, and as expected, appeared normal by aCGH due to data normalization. We also performed an unsupervised analysis of the data, which identified 49 genes with DNA copy number decreases. Both methods identified copy number decreases at 13q14 commonly affected in MM and MGUS patients and thought to harbor a relevant tumor suppressor. Three of the 4 patients with interstitial deletions at 13q14 had striking regions of DNA copy loss whose minimally deleted region was defined by a patient with a small deletion spanning exon 20 of RB1, encoding part of the functionally important ‘pocket domain’ responsible for binding E2F transcription factors. We found RB1 protein levels in MM cell lines correlated with RB1 genomic copy number, and therefore considered the model that RB1 haploinsufficiency contributes to MM. However, we found Rb1 heterozygous (HET) and wild type (WT) mice had indistinguishable steady-state B, T and myeloid compartments in addition to plasma cell induction in response to sheep red blood cell stimulation. Disease burden was similar in HET vs. WT Rb1 mice in a model of NRAS induced tumorigenesis. These results suggest other genomic events cooperate with RB1 copy number loss in MM. Unexpectedly, we found the 3 patients that had an interstitial deletion of RB1 at 13q14 concomitantly harbored a separate interstitial loss at 13q13. Every patient with DNA copy number loss of RB1 also had DNA copy loss within 13q13 (5 patients who lost the entire chromosome and 3 patients with interstitial deletions). The minimally deleted region at 13q13 mapped to the 5′ end of Neurobeachin (NBEA), which encodes a Protein Kinase A (PKA) anchoring protein. We detected NBEA transcripts at low levels in normal human plasma cells. NBEA transcripts and protein were robustly expressed in 3/5 MM cell lines. This is the first report of coordinate copy number loss of RB1 and NBEA on chromosome 13 in MM. Taken together, our data suggest that chromosome 13 deletions in MM may target protein dose level of RB1 and at least one other gene, likely NBEA. Our data provide a novel rationale for future studies to examine the biological consequences of coordinate loss of NBEA and RB1.

A Next Generation Sequencing-Based Approach to Detect Gene Mutations, Copy Number Changes and IGH Translocations in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3364-3364 ◽  
Author(s):  
Niccolo Bolli ◽  
Graham R Bignell ◽  
Peter Ganly ◽  
Elli Papaemmanuil ◽  
Herve Avet-Loiseau ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Copy Number ◽  
Residual Disease ◽  
Gene Mutations ◽  
Myeloma Cell ◽  
Equity Ownership ◽  
Copy Number Changes ◽  
Minimal Residual

Abstract The standard approach to multiple myeloma relies on marrow morphology, serum and urine biochemistry and skeletal imaging for diagnosis. Conventional karyotyping and FISH are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrent mutated genes have been described, some of which are clinically actionable, but their prognostic value remains to be prospectively investigated. Therefore, strategies to investigate the landscape of chromosomal and gene lesions of a large number of myeloma samples in a robust fashion will soon be needed. In this study, we developed a target-enrichment strategy to streamline simultaneous analysis of gene mutations, copy number changes and IGH translocations in multiple myeloma in a high-throughput fashion. We designed Agilent SureSelect cRNA pull down baits to target 246 genes implicated in myeloma and/or cancer, 2538 single nucleotide polymorphisms to detect copy number and allelic ratio at the single-gene and whole-genome level, and we tiled the whole IGH locus to detect IGH translocations and V(D)J rearrangements. As a pilot, we sequenced 13 myeloma cell lines and 10 control haematopoietic cells lines in HiSeq2000 with 75-bp paired end protocol and used standard algorithms developed at the Sanger Institute for analysis. With a mean coverage of the target region of 130x we identified 530 likely oncogenic substitutions and indels, 98.7% of which were validated as real by independent whole exome sequencing. Mutation spectrum of myeloma cell lines was different from patient samples, with mutations in PCLO being the most frequent (8/13) followed by TP53 (7/13), NEB and LTB (6/13). Other genes canonically mutated in myeloma (KRAS, NRAS, BRAF, DIS3, FAM46C) were found at lower frequencies. As expected in a cell line study, most mutations were fully clonal. Using normalized coverage data we were able to study genes and regions showing recurrent copy number changes. With this approach we identified losses in CDKN2C, FAM46C, BIRC2, BIRC3, RB1, DIS3, TRAF3, CYLD and TP53. Furthermore, we could detect deletions in 1p32.3, 1p12, 12p13.31 16q12 and 17p13, as well as amplifications in 1q21.1 and 1q23.3. Validation by SNP-array confirmed an overall positive predictive value of 98% for sequencing data with regards to gain and losses of genes and small chromosomal regions. Analysis of read pairs where mates are aligned to different chromosomes allowed identification and near-bp mapping of the t(11;14) and t(8;14) translocations in 3 cases each, and t(4;14) in two cases, always consistent with the published karyotype for the cell line. No events were found in control cell lines, but our algorithm missed a t(4;14) and a cryptic t(11;14) in two cell lines. Last, we identified reads spanning the V(D)J rearrangement site in most myeloma cell lines, and we are developing tools to reconstruct the tumor-specific rearrangement sequence that could be employed in minimal residual disease monitoring in the future. This panel is now being applied to a cohort of 500 myeloma patient samples with clinical annotation for prognostication. In conclusion, we describe sequencing techniques and analysis tools that can be easily implemented in diagnostic and research laboratories and can be deployed in the study of myeloma pathogenesis, diagnosis, prognosis and as minimal residual disease detection. Disclosures Ganly: Medipics: Equity Ownership; Bone Marrow Cancer Trust, New Zealand: Membership on an entity's Board of Directors or advisory committees. Campbell:14M Genomics Limited: Consultancy, Equity Ownership.

Abstract 2351: MYC gene copy number determine MYC expression and sensitivity to a MYC-inhibitor in multiple myeloma cells

2014 ◽  
Author(s):  
Toril Holien ◽  
Kristine Misund ◽  
Glenn Buene ◽  
Oddrun E. Olsen ◽  
Katarzyna A. Baranowska ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Myeloma Cells ◽  
Myc Gene
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