Comprehensive Ultra High Resolution Analysis of Copy Number and Allele Status Including Novel Microdeletions Spanning NF1 in Acute Myelogenous Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3107-3107
Author(s):  
Sami Malek ◽  
Peter Ouillette ◽  
Whitney Wright ◽  
Amanda Dressel ◽  
Judy Karp ◽  
...  
Keyword(s):  
Copy Number ◽  
Mutational Analysis ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Ras Pathway ◽  
Genomic Changes ◽  
Large Deletions ◽  
Allele Status ◽  
High Resolution Analysis ◽  
Copy Number Changes

Abstract Karyotypic abnormalities are of dominant importance in AML risk prognostication and therapy selection. A comprehensive description of subchromosomal genomic copy number changes and allele status together with gene mutation analysis and identification of chromosomal translocations is needed to fully harvest the prognostic and biological power of genomic changes in AML. We have analyzed DNA from 96 AML-derived pure blast populations (purified using column-based multi parameter negative selection followed by multi-gated FACS sorting) compared with paired buccal DNA using the Affymetrix 6.0 SNP platform. To support data analysis and display, we have developed the software tools PLUT and LOH tool version 2 and have refined dChipSNP. Data were supplemented with blast karyotypes and mutation status of Flt3, NPM1, p53, N-ras and K-ras. Results: AML cases carried between zero and thirty-four subchromosomal losses and gains. Approximately 23% of all cases had ≥3 subchromosomal lesions and approximately 50% of all cases had no such lesions. Of the 22 cases with complex and hypercomplex genomic changes by SNP profiling, ~50% had mutations in p53 exons 5–9 and ~50% were p53 wild-type by sequence analysis. Monoallelic deletion of p53 as part of various deletions at 17p was found in 7 of 96 (7%) cases. Importantly, 7/96=7% additional cases demonstrated UPD at 17p which spanned the p53 locus. Of all 14/96=15% of cases with LOH at 17p (with and without copy loss) 9/14=64% carried p53 mutations and 11/14=79% had complex karyotypes. Of the AML cases with NPM1 mutations (19 of 96 cases or 20%), 75% carried no detectable subchromosomal lesions, while 25% carried between 1 and 4 such lesions. Previously unidentified microdeletions resulting in monoallelic NF1 loss were identified on 17p in a total of 10 of 96 (10%) cases, thus suggesting activation of the Ras pathway independent of Ras mutations (all affected cases had wild-type N-ras and K-ras) in a substantial subset of AML cases. Mutational analysis of all coding exons of NF1 on the retained allele in the affected cases is ongoing. Additional recurrent microdeletions were identified on chromosomes 3p and 3q as well as 12p, the latter encompassing the genes ETV6 and p27, as previously reported. Finally, analysis of the prognostically and therapeutically important deletions 5q and 7q identified large deletions without recurrent microdeletions. Regions of minimal loss on 5q have been delineated.

scholarly journals RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (2) ◽  
pp. 758-766 ◽  
Author(s):  
R Ruggieri ◽  
A Bender ◽  
Y Matsui ◽  
S Powers ◽  
Y Takai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Copy Number ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Wild Type ◽  
Ras Gene ◽  
Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

scholarly journals High-Resolution Analysis of Genomic Copy Number Changes

Genomics ◽  
2010 ◽  
pp. 1-31 ◽  
Author(s):  
Mario Hermsen ◽  
Jordy Coffa ◽  
Bauke Ylstra ◽  
Gerrit Meijer ◽  
Hans Morreau ◽  
...  
Keyword(s):  
High Resolution ◽  
Copy Number ◽  
Genomic Copy Number ◽  
Genomic Copy ◽  
High Resolution Analysis ◽  
Resolution Analysis ◽  
Copy Number Changes

scholarly journals Telomerase-Mediated Repair of Induced DNA Breaks Leads to Increased Genomic Instability in Multiple Myeloma Cells: Possible Mechanism and Translational Significance

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5170-5170
Author(s):  
Jagannath Pal ◽  
Masood Shammas ◽  
Subodh Kumar ◽  
Maria Gkotzamanidou ◽  
Puru Nanjappa ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Genomic Instability ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Chemotherapeutic Agents ◽  
Telomere Maintenance ◽  
Dna Breaks ◽  
Genomic Changes ◽  
Time Points ◽  
Copy Number Changes

Abstract Telomerase has multiple functions. We here reported that besides telomere maintenance, telomerase is also involved in the repair of DNA breaks in myeloma (MM) cells. By sequence analyses of “TTAGGG” enriched genomic fragments, we also mapped the interstitial telomeric repeat sequences (ITS) and demonstrate that telomerase mediated repair involves the insertion of ITS within cancer genome. More importantly, we show that such insertions which occur at higher frequency in cancer vs. normal cells, are reduced in the presence of telomerase inhibitor (TI). We hypothesized that if the ITS if inserted in large numbers following massive DNA breaks, induced by a genotoxic agent, could become substrates for recombination (which is elevated in myeloma) leading to increased genomic instability. To investigate if repair of induced DNA breaks by telomerase would contribute to genomic instability, DNA breaks in RPMI cells were induced by UV, cells cultured in the presence of TI or control oligo for four 4 days, drug was then withdrawn and cells continued in culture for 30 days. Cells were harvested at days 10, 20 and 30 and their genomic DNA evaluated for copy number changes using SNP6.0 arrays. Genome of day 10 for each sample was used as baseline to identify genomic changes in corresponding day 20 and day 30 samples. We observed a gradual increase in deletion events in UV treated control cells from 14-fold at day 20 to 147-fold at day 30, relative to corresponding changes in UV-TI samples at respective time points. Likewise, the amplifications were massively increased (by 500-fold) at day 30, relative to changes in UV-TI cells at the respective time points. When we compared the fold changes in copy number in these samples relative to UV unexposed baseline control cells, changes in TI-treated cells remained close to the baseline till day 30. However, there was a sharp rise in copy number changes in UV treated control cells at corresponding time point. These data indicate that a brief telomerase inhibition during induced DNA damage may prevent late genomic instability. We have further evaluated the translational significance of these findings. We investigated if inhibition of telomerase mediated repair could sensitize cancer cells to DNA damaging chemotherapeutic agents. RPMI MM cells, pretreated with TI or control oligo for 60 hours, were incubated with melphalan at various concentrations for 48 hrs and cell viability assessed. Cell death in cells pretreated with TI was significantly higher than control cells at each dose level suggesting that TI pretreatment sensitizes cells to DNA damaging agents. Telomerase mediated repair involving insertion of telomeric repeats at interstitial break sites prevents immediate genomic loss, and inhibition of this repair sensitizes MM calls to DNA damaging agent. We conclude that presence of a TI during treatment with a DNA damaging agent would not only enhance the efficacy of treatment but may also protect cancer cells against therapy induced late genomic instability and evolution of aggressive malignant clones. Disclosures No relevant conflicts of interest to declare.

scholarly journals RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (2) ◽  
pp. 758-766
Author(s):  
R Ruggieri ◽  
A Bender ◽  
Y Matsui ◽  
S Powers ◽  
Y Takai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Copy Number ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Dominant Negative ◽  
Yeast Cells ◽  
Wild Type ◽  
Ras Gene ◽  
Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

Mechanism of Altered Nucleo-Cytoplasmic Traffic of Nucleophosmin in Acute Myelogenous Leukemia Carrying Exon-12 NPM Mutations (NPMc+ AML).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4396-4396
Author(s):  
Brunangelo Falini ◽  
Niccolo Bolli ◽  
Jing Shan ◽  
Maria Paola Martelli ◽  
Ildo Nicoletti ◽  
...  
Keyword(s):  
Acute Myelogenous Leukemia ◽  
Nuclear Export ◽  
Mutational Analysis ◽  
Nuclear Export Signal ◽  
Normal Karyotype ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Nuclear Localization Signals ◽  
Tryptophan Residues ◽  
Acute Myelogenous

Abstract We recently found that about one-third of adult AML (60% of all AML with normal karyotype) display aberrant cytoplasmic expression of nucleophosmin (NPM) which is due to mutations occurring at the exon-12 of NPM gene (Falini B, et al., Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype..N Engl J Med2005; 352: 254–266). Hereby, we clarify the molecular mechanism underlying cytoplasmic NPM accumulation that yet remained to be elucidated. AML-associated mutated NPM alleles encode abnormal NPM proteins (25 mutants so far identified) which have acquired at the C-terminus a nuclear export signal-(NES) motif and lost at least one of tryptophan residues 288 and 290 which determine nucleolar localization. Both alterations are crucial for mutant NPM export from nucleus to cytoplasm. In fact, the cytoplasmic NPM accumulation is blocked by leptomycin-B and ratjadones which are specific inhibitors of exportin-1/CRM1, and by re-insertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. Thus, for cytoplasmic accumulation of NPM to occur, the NES motif and tryptophan mutations must act in concert. Possibly, when NPM mutans enter the nucleus by virtue of their nuclear localization signals (NLS), their capability to bind nucleoli must be hindered at least partially to become a CRM-1 target. Specific antibodies anti-NPM mutant proteins showed that the mutants localized exclusively in the cytoplasm and recruited in that site the wild-type NPM protein which is physiologically located in the nucleoli. These findings suggest that the NPM mutants may interfere with the functions of wild-type NPM and possibly contribute to leukemogenesis. Immunostaining of 393 AML cases using anti-NPM monoclonal antibodies predicted the presence of NPM exon-12 mutations in all 191 NPM-cytoplasmic positive cases. This finding is consistent with the fact that, despite genomic heterogeneity, all NPM mutants contain a NES motif which, in the presence of altered tryptophans, promotes their rapid export from the nucleus to the cytoplasm. The immunohistochemical test is diagnostically relevant since it can be used as simple first-step procedure in molecular-genetic characterization of AML and as a surrogate for mutational analysis in selected cases. These findings are also clinically relevant since cytoplasmic NPM/NPM mutations are predictors of good response to induction therapy and favourable prognosis in AML with a normal karyotype.

Genome-Wide Analysis of Microdeletions and Identification of NF1 Inactivation in Acute Myelogenous Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 165-165
Author(s):  
Sami Malek ◽  
Peter Ouillette ◽  
Yin Wang ◽  
Yan Liu ◽  
Whitney Wright ◽  
...  
Keyword(s):  
Acute Myelogenous Leukemia ◽  
Copy Number ◽  
Research Funding ◽  
Normal Karyotype ◽  
B Cell Lymphoma ◽  
Myelogenous Leukemia ◽  
Acute Myelogenous ◽  
Copy Number Changes ◽  
Induced Apoptosis ◽  
High Level

Abstract Abstract 165 Genomic aberrations are of dominant importance to the biology and clinical outcome of patients with acute myelogenous leukemia (AML). To further our understanding of such aberrations in AML, we analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for subchromosomal copy number changes and allele identities using ultra-high-density Affymetrix SNP 6.0 array-based genomic profiling. A total of 358 somatically acquired copy number changes were detected in 95 AML genomes. We detected 16 losses and 22 gains of entire chromosomes, 285 subchromosomal losses and 35 subchromosomal gains. No recurrent high-level amplifications or recurrent homozygous deletions were identified. Eight of the 34 AML cases (24%) with normal karyotype each had one lesion detected through 6.0 array profiling, all but one of which was less than 4Mb in length. Focusing on microdeletions as potential indicators of the locations of novel tumor suppressor genes or genes with importance to AML biology, we identified 60 deletions that were less than 1 Mb in length and 158 deletions of less than 5 Mb, the vast majority of which were undetectable by conventional cytogenetics. Through fine mapping of microdeletions on 17q, we identified Neurofibromin 1 (NF1) null states due to mutations or absent expression in ∼7% of AML. NF1 mutations were present in the hematopoetic stem cell compartment (CD34+/CD38- cell population) and siRNA-mediated NF1 suppression using recombinant lentiviruses significantly increased colony formation of primary AML blasts in methylcellulose. Further, AML blasts without functional NF1 displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mTOR signaling for survival. As an additional validation of using microdeletions to guide pathogenetic gene discovery, we identified deletions involving RUNX1, IRF8, Core Binding Factor Beta (CBFB) and Casitas B-cell lymphoma B (CBLB), genes known to be altered in AML. IRF8 expression was found to be absent in ∼30% of all AML but sequencing of all coding exons of IRF8 of 48 AML cases did not disclose somatically acquired mutations. In summary, this comprehensive description of subchromosomal copy number changes and microdeletions in adult AML substantially adds to our knowledge of the pathological anatomy of the AML genome and should inform future searches for novel genes with importance to AML biology. Disclosures: Malek: Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding. Erba:Lilly: Research Funding; Antisoma: Research Funding; Wyeth: Research Funding; Cephalon: Honoraria, Research Funding; MGI Pharma: Honoraria; Pharmion: Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gemin-X: Research Funding; Kanisa: Research Funding.

Evolution of Genomic Changes and Their Significance in Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 605-605
Author(s):  
Masood A Shammas ◽  
Herve Loiseau ◽  
Cheng Li ◽  
Leutz Buon ◽  
Samir B Amin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Clinical Outcome ◽  
Genomic Instability ◽  
Copy Number ◽  
Research Funding ◽  
Chromosome 1 ◽  
Poor Clinical Outcome ◽  
Genomic Changes ◽  
Aggressive Phenotype ◽  
Copy Number Changes

Abstract Abstract 605 Despite therapeutic advances and high response rates, most if not all patients with multiple myeloma (MM) develop drug resistance and relapse and curative outcomes remain elusive. A prominent feature of MM is striking genomic instability that evolves with the progression of disease. This genomic instability is considered responsible for development of aggressive phenotype associated with relapsed disease and for development of drug resistance. The molecular basis for the generation of this genetic diversity in cancer cells thus has important implication in understanding cancer progression and therapy. However the genomic evolution in MM patient samples has not been documented. Here, we have utilized single nucleotide polymorphism (SNP) arrays to monitor genome-wide changes in heterozygosity and copy number, in two CD138+ MM cell samples collected 6 months apart from 14 MM patients. Genomic changes acquired in the late tumor samples were identified using early samples as baseline. We defined an event as detectable change in heterozygosity/copy number in three or more consecutive SNPs. All 14 MM patients acquired new genomic change in the later sample at a frequency ranging from 0.021 - 2.674 % (i.e. per 100 informative loci investigated). Although the rate of mutation varied, 12 out of 14 patients had acquired >100 mutational events. Chromosomes 1, 13, and × showed large areas of copy number change in several patients. We also evaluated if genomic changes correlate with changes in expression of corresponding genes. Selecting larger areas of genome, we observed that copy number changes correlate well with the change in expression of genes in these areas. As expected, we also observed a correlation among changes in copy number, heterozygosity, and gene expression at several chromosomal loci. In a number of instances frequently recurrent changes were observed. For example, recurrent copy number changes in areas spanning 1q42.13-1q44 and 1p12-1p12 of chromosome 1 were seen in majority (12 out of 14 and 13 out of 14) patients, whereas copy number changes in the p arm of chromosome × were present in all patients. Similarly the region of chromosome × spanning xq42.13-xq44 showed change in heterozygosity in majority of patients. We also observed that some of the newly acquired changes in late samples correlated with genomic markers of poor clinical outcome. We evaluated prognostic significance of these changes in 192 uniformly treated patients with MM with genomic gains and losses data from SNP array and survival information. Changes in chromosomal regions 1p12 and xp22.1-xp22.33 frequently observed in late samples were significantly (p = 0.017 and 0.037) associated with poor survival in these patients. These data suggest that MM cells acquire changes associated with aggressive phenotype and shorter survival. In conclusion we observe that MM patients acquire genomic changes at a very high rate; and certain chromosomal regions are more vulnerable predicting poor clinical outcome. These data also suggest a need to target mechanisms mediating genomic instability for therapeutic application. Disclosures: Richardson: Keryx Biopharmaceuticals: Honoraria. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Dissecting the genomic landscape of metastatic bladder cancer using circulating tumor DNA.

2017 ◽  
Vol 35 (6_suppl) ◽  
pp. 378-378
Author(s):  
Werner J. Struss ◽  
Gillian Vandekerkhove ◽  
Matti Annala ◽  
Tilman Todenhoefer ◽  
Kevin Beja ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Peripheral Blood ◽  
Copy Number ◽  
Mononuclear Cells ◽  
Blood Stream ◽  
Copy Number Variations ◽  
Genomic Alterations ◽  
Peripheral Blood Mononuclear ◽  
Genomic Changes ◽  
Copy Number Changes

378 Background: To date, only limited data are available on genomic alterations present in metastatic lesions of patients with metastatic urothelial carcinoma (mUC). The constant release of cell free DNA (cfDNA) from tumor cells into the peripheral blood stream may provide a valuable source for detection of cancer associated somatic copy number variations and mutations. The aim of this project was to evaluate the feasibility of detecting clinically relevant genomic alterations in mUC by next-generation sequencing (NGS) of circulating cfDNA. Methods: Peripheral blood (20ml) was collected from 15 patients with mUC before chemotherapy. Plasma cfDNA was isolated using the QIAAmp Circulating Nucleic Acid Kit. Germline control DNA was extracted from peripheral blood mononuclear cells (PBMCs) using the DNEasy Kit (Qiagen). We performed deep (>500X) targeted DNA sequencing on cfDNA and germline DNA using a custom NimbleGen SeqCap EZ Choice Library and Illumina sequencers. Our custom panel included 50 bladder cancer-relevant genes, including key tumor suppressors (e.g. TP53, RB1, CDKN2A), cell cycle drivers (e.g. CCND1, CCNE1), DNA repair genes (e.g. ATM, BAP1, ERCC2), PI3K pathway genes (e.g. PIK3CA, PTEN, AKT1), and other oncogenic pathways (e.g. RAS/RAF, EGFR, ERBB2, PPARG, FGFR3). Results: Somatic mutations and/or copy number changes were detected in 14/15 samples, with a median variant allele frequency >5%. The overall mutational landscape was highly consistent with bladder cancer, including mutations in TP53 and ARID1A (and other chromatin modifiers), as well as hotspot activating mutations in PIK3CA. Typical copy number changes, including focal amplifications of ERBB2, KRAS, and CCNE1 were detected. We identified complex gene rearrangements, including an activating FGFR3 fusion in one case. Extended analyses with a larger patient population are currently pending. Conclusions: Non-invasive characterization of genomic changes in peripheral blood of patients with mUC using NGS of plasma cfDNA is feasible in a high proportion of patients. Analysis of cfDNA might therefore provide an improved understanding of genomic changes in mUC and could be developed as a biomarker to help guide the use of targeted therapies.

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