Status of Chromosome 13 in Multiple Myeloma: Integrated Approach Using SNP Mapping Array and Gene Expression Array.

Abstract Deletions on chromosome 13 are thought to be one of the most important prognostic features in Multiple Myeloma (MM). The biology underlying this is, however, uncertain. Chromosome 13 abnormalities have been evaluated conventionally by FISH using probes for 13q14, covering the retinoblastoma gene (RB1) region. Typically, for recurrent regions of loss of heterozygosity (LOH) it is possible to map a minimally deleted region within which an important gene may be located. This should be the case with 13q−, or alternatively there may be linkage with another genetic lesion, which could be contributing to the poor prognosis. Following the implementation of high-density single-nucleotide polymorphism (SNP) array, it is now possible to genotype the whole human genome with a mapping resolution of less than 50 Kb. Thus, the SNP array approach offers an opportunity to analyze both copy number abnormalities and LOH simultaneously. The aim of this study was to determine the numerical alterations, LOH and changes in the gene expression profile of the chromosome 13 in MM, and its possible association with other genetic events. For this purpose, we analyzed 17 patients included on the Myeloma IX trial with deletion of 13q14 compared with 22 samples without deletion, using Affymetrix 50K SNP arrays and Affymetrix U133 Plus 2 expression array. IGH translocations and 13q deletion were determined by FISH. dChipSNP and WGSA programs were used to analyze the data. With respect to 13q14, there was 100% correlation between FISH and SNP array results. 16 out of 17 cases with deletion of the RB1 gene by FISH analysis showed loss of 13q arm by SNP array, demonstrating that loss of the whole chromosome 13 is responsible for 13q deletions found in MM in >90% of cases, with only one case showing a defined region of deletion of chromosome 13 (13q14.11–13q21.2). Using gene expression arrays we could not define a specific pattern characteristic of expression loss in genes at 13q. Lower RB1 expression levels were not only restricted to cases with del(13). However, samples containing IGH translocations (t(11;14) and t(4;14)) without del(13) showed up to 4 times more RB1 expression, suggesting that MM evolution in cases containing IGH translocations is independent of RB1 expression. Interestingly, the hyperdiploid cases with and without del(13) expressed similar level of RB1. We also investigated whether other key cell cycle regulatory genes were associated with del(13); in particular, 4 cases showed 9p21 LOH by SNP array and no different gene expression levels, which suggest that LOH does not seem to be a mechanism of lost of expression of CDKN2A, CDKN2B and p14/ARF. We could not find any significant correlation with del(13) and expression of cell cycle regulatory genes, apart from 8/17 samples with del(13) that had low expression of p53 gene, including 6 t(4;14) cases and 2 t(11;14) cases. Also, 2 cases without monosomy 13 (1 with t(4;14) and 1 with t(11;14)), showed low p53 expression levels. However, SNP array data did not show any deletion at 17p in 38 cases, with the exception of a case with monosomy 13 and t(11;14) in which SNP array data showed loss at 17pter-17q21.2 and FISH detected p53 deletion. Further investigation between the association of p53 and del(13) are ongoing and maybe useful in defining the biology of this poor subgroup of patients.

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Continuous absence of metaphase-defined cytogenetic abnormalities, especially of chromosome 13 and hypodiploidy, ensures long-term survival in multiple myeloma treated with Total Therapy I: interpretation in the context of global gene expression

Blood ◽

10.1182/blood-2002-09-2873 ◽

2003 ◽

Vol 101 (10) ◽

pp. 3849-3856 ◽

Cited By ~ 101

Author(s):

John Shaughnessy ◽

Joth Jacobson ◽

Jeff Sawyer ◽

Jason McCoy ◽

Athanasios Fassas ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Multiple Myeloma ◽

Global Gene Expression ◽

Cell Cycle Gene ◽

Term Survival ◽

Cytogenetic Abnormalities ◽

Chromosome 13 ◽

Prognostic Implications ◽

Total Therapy

Abstract Metaphase cytogenetic abnormalities (CAs), especially of chromosome 13 (CA 13), confer a grave prognosis in multiple myeloma even with tandem autotransplantations as applied in Total Therapy I, which enrolled 231 patients between 1989 and 1994. With a median follow-up of almost 9 years, the prognostic implications of all individual CAs, detected prior to treatment and at relapse, were investigated. Among all CAs and standard prognostic factors examined prior to therapy, only hypodiploidy and CA 13 (hypo–13 CA), alone or in combination, were associated with shortest event-free survival and overall survival (OS). The shortest postrelapse OS was observed with hypo–13 CA, which was newly detected in 18 of all 28 patients presenting with this abnormality at relapse. Superior prognosis was associated with the absence of any CA at both diagnosis and relapse (10-year OS, 40%). The lack of independent prognostic implications of other CAs points to a uniquely aggressive behavior of hypo–13 CA (present in 16% of patients at diagnosis). With the use of microarray data in 146 patients enrolled in Total Therapy II, overexpression of cell cycle genes distinguished CA from no CA, especially in cases of del(13) detected by interphase fluorescence in situ hybridization (FISH). FISH 13, resulting in a haploinsufficiency of RB1 and other genes mapping to chromosome 13, as well as activation of IGF1R, appears to have an amplifying effect on cell cycle gene expression, thus providing a molecular explanation for the dire outcome of patients with CA 13 compared with those with other CAs.

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Interactive Effects of HDAC Inhibitors and TRAIL on Apoptosis Are Associated with Changes in Mitochondrial Functions and Expressions of Cell Cycle Regulatory Genes in Multiple Myeloma

Neoplasia ◽

10.1593/neo.04655 ◽

2005 ◽

Vol 7 (7) ◽

pp. 646-657 ◽

Cited By ~ 120

Author(s):

Tamer E. Fandy ◽

Sharmila Shankar ◽

Douglas D. Ross ◽

Edward Sausville ◽

Rakesh K. Srivastava

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Hdac Inhibitors ◽

Regulatory Genes ◽

Interactive Effects ◽

Mitochondrial Functions ◽

Cell Cycle Regulatory Genes

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Genome-Wide Analyses Identify Recurrent Amplifications of Receptor Tyrosine Kinases and Cell-Cycle Regulatory Genes in Diffuse Intrinsic Pontine Glioma

Journal of Clinical Oncology ◽

10.1200/jco.2011.35.5677 ◽

2011 ◽

Vol 29 (30) ◽

pp. 3999-4006 ◽

Cited By ~ 194

Author(s):

Barbara S. Paugh ◽

Alberto Broniscer ◽

Chunxu Qu ◽

Claudia P. Miller ◽

Junyuan Zhang ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Tyrosine Kinases ◽

Receptor Tyrosine Kinases ◽

Diffuse Intrinsic Pontine Glioma ◽

Regulatory Genes ◽

Low Grade ◽

Pontine Glioma ◽

Gene Expression Signatures ◽

Cell Cycle Regulatory Genes

Purpose Long-term survival for children with diffuse intrinsic pontine glioma (DIPG) is less than 10%, and new therapeutic targets are urgently required. We evaluated a large cohort of DIPGs to identify recurrent genomic abnormalities and gene expression signatures underlying DIPG. Patients and Methods Single-nucleotide polymorphism arrays were used to compare the frequencies of genomic copy number abnormalities in 43 DIPGs and eight low-grade brainstem gliomas with data from adult and pediatric (non-DIPG) glioblastomas, and expression profiles were evaluated using gene expression arrays for 27 DIPGs, six low-grade brainstem gliomas, and 66 nonbrainstem low-grade gliomas. Results Frequencies of specific large-scale and focal imbalances varied significantly between DIPGs and nonbrainstem pediatric glioblastomas. Focal amplifications of genes within the receptor tyrosine kinase–Ras–phosphoinositide 3-kinase signaling pathway were found in 47% of DIPGs, the most common of which involved PDGFRA and MET. Thirty percent of DIPGs contained focal amplifications of cell-cycle regulatory genes controlling retinoblastoma protein (RB) phosphorylation, and 21% had concurrent amplification of genes from both pathways. Some tumors showed heterogeneity in amplification patterns. DIPGs showed distinct gene expression signatures related to developmental processes compared with nonbrainstem pediatric high-grade gliomas, whereas expression signatures of low-grade brainstem and nonbrainstem gliomas were similar. Conclusion DIPGs comprise a molecularly related but distinct subgroup of pediatric gliomas. Genomic studies suggest that targeted inhibition of receptor tyrosine kinases and RB regulatory proteins may be useful therapies for DIPG.

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A Comparative Analysis of Chromosome 13 Gene Expression and Copy Number Changes in Multiple Myeloma Using Array-Based Methods.

Blood ◽

10.1182/blood.v106.11.1557.1557 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1557-1557

Author(s):

Angela S. Baker ◽

Tae-Hoon Chung ◽

Tyler S. Pidgeon ◽

Catherine Mancini ◽

Tammy Price-Troska ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Tumor Suppressor ◽

Copy Number ◽

Loss Of Function ◽

Chromosome 13 ◽

Expression Levels ◽

Genomic Copy Number ◽

Genomic Copy ◽

Copy Number Changes

Abstract Chromosome 13 (Δ13) abnormalities are found in greater than 50% of patients with Multiple Myeloma (MM). MM is most commonly defined by chromosome 13 monosomy or 13q loss (85%). Interstitial deletions comprise the remaining 15%. Many studies have revealed that Δ13 in MM are associated with poor survival and reduced response to therapy. Genes mapping to chromosome 13 may be involved in pathogenesis and/or progression of the disease due to loss of function from gene mutation or from epigenetic effects such as haploinsufficiency. In this study, array-based comparative genomic hybridization coupled with microarray technology (aCGH) is used to detect gene copy number loss on chromosome 13 from nine MM patient samples. Whole genome long-oligo microarrays constructed by Agilent Technologies were used which contain 40,000 genes that span the human genome with an average spatial resolution of ~75 kb. Using genomic DNA isolated from MM patients with interstitial deletions on chromosome 13, DNA was amplified, labeled and hybridized with a differentially labeled normal DNA reference to determine gene/genomic copy number changes. Arrays were analyzed to search for the minimum region of loss based upon single copy loss for a series of nearby mapping transcripts. A common region of loss of 2.2 Mb, at 13q14.2 was detected. Additionally, we investigated the correlation between genomic copy number change and the expression level for MM patients in the13q region. From an independent gene expression data set whose expression measurements were conducted with Affymetrix HG-U133A v2 microarrays, data was selected that corresponded to the samples used for current aCGH. Expression values from MM samples were divided by the mean expression values from 12 normal bone marrow samples for each gene and the resulting values were treated as surrogate ratios between MM and normal samples. Probes from both microarrays were then aligned according to their chromosomal positions and merged if their chromosomal positions overlapped. Composite chromosomal maps were generated that displayed the expression levels and copy number changes. The maps were used to differentiate chromosomal regions in 13q where copy number changes and expression levels show high correlation and regions where such correlation was not observed. Although the number of probes sampled in the expression microarray was much smaller than those in aCGH microarray, a chromosomal region of great interest, that encompasses 13q14.2, arose naturally from this analysis. Although the mechanism by which loss of 13q effects tumorigenesis in MM could be a haploinsufficiency model, we are not ruling out the presence of a tumor suppressor gene in this region. We are evaluating candidate tumor suppressor genes in the region for loss of function by mutational analysis and hypermethylation studies.

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Pegylated G-CSF Mobilizes CD34+ Cells with Different Stem and Progenitor Cell Subsets and Distinct Functional Properties in Comparison with Unconjugated G-CSF.

Blood ◽

10.1182/blood.v108.11.3382.3382 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3382-3382

Author(s):

Ingmar Bruns ◽

Ulrich Steidl ◽

Johannes C. Fischer ◽

Sascha Raschke ◽

Guido Kobbe ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Multiple Myeloma ◽

Stem Cell ◽

Functional Properties ◽

Peripheral Blood ◽

Single Injection ◽

Expression Levels ◽

Biological Studies ◽

Cd34 Cells

Abstract Current regimens for peripheral blood stem cell (PBSC) mobilization in patients with multiple myeloma are based on daily subcutaneous injections of G-CSF starting shortly after cytotoxic therapy. Recently a polyethylenglycole (PEG)-conjugated G-CSF has been introduced which has a substantially longer half-life than the original formula and therefore provides the basis for long-lasting G-CSF serum-levels after a single injection. In this study, we compared gene expression patterns, subset composition and functional properties of CD34+ cells and highly purified HSC mobilized with cyclophosphamide and either Peg-G-CSF or G-CSF. Cells were derived from peripheral blood of patients with multiple myeloma. After the end of chemotherapy, 7 patients got a single injection of Peg-G-CSF whereas 9 patients received daily G-CSF resulting in an equal cumulative dose. Gene expression analysis was performed using Affymetrix HG Focus GeneChips. Key functional genes were verified by RT-PCR. Subset analysis and fluorescence based cell sorting has been conducted to assess the effects of stimulation with either pegylated or unconjugated G-CSF on CD34+ subset composition and to obtain HSCs. Cell cycle and apoptosis assays as well as clonogenic assays were for functional corroboration. The same patients with multiple myeloma who had donated CD34+ cells for the molecular and biological studies were transplantated with Peg-G-CSF- or G-CSF-mobilized PBSC. Peg-G-CSF-mobilized cells showed lower expression of genes characteristic for erythroid and later stages of myeloid differentiation as well as a lower BFU-E/CFU-GM ratio compared to G-CSF-mobilized cells. In turn, we found higher expression levels of genes indicative of early hematopoiesis including HOXA9, MEIS1, MLL and GATA3. Subset analyses revealed a greater number of HSC and CMP (common myeloid progenitors) and a lower number of MEP (megakaryocyte-erthrocyte progenitors) in Peg-G-CSF-mobilized CD34+ cells. Cell cycle-promoting genes including cyclins and kinases were higher expressed in Peg-G-CSF-mobilized cells. On the other hand human HTm4, which causes cell cycle arrest in hematopoietic cells, was lower expressed compared to G-CSF-mobilized cells. This is emphasized by a significant higher proportion of actively cycling CD34+ cells after pegfilgrastim-mobilization. Higher gene expression levels of HOXA9, MEIS1 and GATA3 were also found in sorted Peg-G-CSF-mobilized HSC in comparison to G-CSF-mobilized HSC. Moreover, Peg-G-CSF-mobilized HSC showed a lower apoptosis rate and a greater proportion of cells in S- and G2/M phase of cell cycle. After transplantation of Peg-G-CSF-mobilized stem- and progenitor cells we observed earlier leukocyte recovery compared to G-CSF-mobilized transplants. Our data demonstrate that Peg-G-CSF and G-CSF stimulation differentially affects the expression of key regulatory genes and functional properties of mobilized HSC as well as their progeny, which might be important for their application in stem cell transplantation.

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