Genetic and Epigenetic Silencing of Mesoderm Specific Transcript (MEST) In Acute Myelogenous Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3639-3639
Author(s):  
Anna R Poetsch ◽  
Rainer Claus ◽  
Lars Bullinger ◽  
Tania Witte ◽  
Michael Lübbert ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Cpg Island ◽  
Normal Karyotype ◽  
Epigenetic Silencing ◽  
Myelogenous Leukemia ◽  
Chromosome 7 ◽  
Special Importance ◽  
Monosomy 7 ◽  
Chromosomal Changes

Abstract Abstract 3639 About 10 % of patients with either myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) present either with deletions on the long arm of chromosome 7 or monosomy 7. These chromosomal aberrations are associated with a poor prognosis. Following biallelic inactivation as proposed by Knudsen, a “second hit” of the remaining allele might be required for loss of gene function. Epigenetic silencing might display such a hit in particular since no additional genetic hits could be identified so far. The role of epigenetic regulation might be of special importance in patients with -7/7q-, since several studies have shown that patients harbouring a chromosome 7 abberrations do more benefit from a therapy with demethylating agents as compared to patients with other chromosomal changes. To address the issue of epigenetic silencing in these AML cases we utilized DNA methylation profiling to identify the potential tumor suppressor genes on chromosome 7. We used MBD2 based enrichment of methylated DNA from 4 AML patient samples with monosomy 7 and from 23 patient samples with with other chromosomal changes including normal karyotype AML. For validation we analyzed regional DNA methylation using quantitative MassArray technology on DNA from 115 del(7q) AML or monosomy 7 patients as well as normal karyotype patients (n=20) and CD34 + cells from healthy individuals (n=5). We could identify the gene MEST to be silenced by hypermethylation (> 30 %) of a CpG island on the remaining allele in 20% of the patients with -7/7q- and 40% of patients with normal karyotype. Thus, preferentially in patients that do not have a deletion already. MEST is an imprinted gene located on 7q32.2. However, silencing is correlated with hypermethylation of a CpG island located at an alternative promoter of MEST, independent of the methylation status of the imprinting control region. In two patient samples with monosomy 7 and hypermethylation of MEST (> 80%), DNA methylation was erased after 5-Aza-2′-deoxycytidine (DAC) treatment to less than 10 %. In concordance, also AML cell lines with hypermethylation of the MEST locus loose methylation after sublethal DAC treatment which leads to a reexpression of the gene. Thus, we hypothesize that MEST functions as a tumor suppressor in AML and is genetically as well as epigenetically silenced AML. Reactivation of MEST by demethylating treatment may contribute to the mechanism by which demethylating drugs display their therapeutic potential in leukemia. Disclosures: No relevant conflicts of interest to declare.

Genetic and Epigenetic Profiling on 7q22 Identifies ATXN7L1 As Candidate Tumor Suppressor in Acute Myelogenous Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1408-1408 ◽  
Author(s):  
Anna R Poetsch ◽  
Rainer Claus ◽  
Jacques Bonnet ◽  
Didier Devys ◽  
Frank G. Rücker ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Conflicts Of Interest ◽  
Cpg Island ◽  
Myelogenous Leukemia ◽  
Chromosome 7 ◽  
Alternative Mechanism ◽  
Saga Complex ◽  
Monosomy 7 ◽  
Cd34 Cells

Abstract Abstract 1408 About 10 % of patients with acute myeloid leukemia (AML) present with either monosomy 7 or deletions on the long arm of chromosome 7. This chromosomal aberration is associated with poor prognosis and adverse therapeutic response. Following biallelic inactivation as proposed by Knudsen's hypothesis, a “second hit” of the remaining allele might be required for loss of gene function. So far, no consistent additional genetic hits could be identified in the minimally deleted regions. Thus, epigenetic silencing might display a local alternative mechanism ultimately causing silencing of a potential tumor suppressor. Based on cytogenetic data we defined a minimally deleted region on 7q22.2 of 2–3 Mb in size to narrow down the location of the putative tumor suppressor. It is flanked by the microsatellite markers D7S1503 and D7S1841. We utilized comprehensive DNA methylation profiling in the CpGrich areas of the minimal deleted regions by high resolution DNA methylation assessment by MassARRAY to identify aberrant epigenetic patterns in these regions. In this region we quantitatively analyzed CpG island methylation in a cohort of 64 AML patients with del(7q), 11 with monosomy 7, ten with normal karyotype and five CD34+ bone marrow cell DNA from healthy donors as controls. We identified four genes (PRES, LHFPL3, ATXN7L1, and CDH28) that are hypermethylated in their promoters in this region with significant mean methylation differences from 5 to 20 % comparing AML to healthy controls. Hypermethylation occurred both in patients with chromosome 7 aberrations and in patients with normal karyotypes. To narrow down aberrantly methylated genes to functional relevant candidates, we excluded those that were not expressed in healthy CD34+ cells. ATXN7L1, a gene located on 7q22.2 and coding for a potential subunit of the histone acetyl transferase (HAT) and deubiquitinase SAGA complex, was expressed in healthy CD34+ cells and granulocytes, but downregulated upon hypermethylation (>30%) in patients. To elucidate a potential functional role of ATXN7L1, we immunopurified SAGA from HeLa cells using antibodies against a SAGA subunit, GCN5,and analyzed the coimmunoprecipated proteins by subsequent mass spectrometry. Based on the identity of the co-precipitated proteins we demonstrate that ATXN7L1 is a bona fide component of the human SAGA complex. Thus our results suggest that ATXNL1 has a similar role as the closely related protein, ATXN7 that is known to be involved in transcription regulation via USP22 dependent deubiquitination of histones. Experiments to understand how ATXN7L1 silencing may affect the leukemic phenotype are in progress. Disclosures: No relevant conflicts of interest to declare.

Protein Tyrosine Phosphatase Receptor Type O (PTPRO) Exhibits Tumor Suppressor Properties in K562 Cells by Dephosphorylating Bcr-Abl.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2914-2914
Author(s):  
Sarmila Majumder ◽  
Tasneem Motiwala ◽  
Kalpana Ghoshal ◽  
Huban Kutay ◽  
Jharna Datta ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Protein Tyrosine Phosphatase ◽  
Cpg Island ◽  
Tyrosine Phosphatase ◽  
Ectopic Expression ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Receptor Type ◽  
Protein Tyrosine ◽  
Catalytically Active

Abstract Regulation of protein phosphorylation by concerted action of protein kinases and phosphatases is important for normal physiological processes. Altered function or expression of one or more components of these regulatory molecules leads to many pathological conditions including cancer. We have previously shown that the truncated form of the receptor-type protein tyrosine phosphatase PTPROt predominantly expressed in haematopoietic cells is suppressed in Chronic Lymphocytic Leukemia (B-CLL). A direct correlation of CpG island methylation and reduced expression of the gene was observed in primary CLL samples and in several leukemia cell lines. To assess the functional significance of loss of PTPROt function in leukemia, we selected K562 cells as a model system, as PTPROt expression is silenced in these cells and is reactivated upon treatment with DNA hypomethylating agents. Ectopic expression of the catalytically active PTPROt inhibited growth of K562 cells and their clonogenic survival in soft agar (a characteristic of cancer cells). Further, cells expressing PTPROt exhibited delayed entry into S-phase from G0/G1 phase. Induction of apoptosis increased significantly in K562 cells expressing functional phosphatase upon serum withdrawal or exposure to the apoptogenic agent camptothecin. Tumorigenic potential of K562 cells in athymic nude mice was also significantly reduced upon ectopic expression of PTPROt. Finally, we demonstrate that the Bcr-Abl fusion protein, product of abnormal chromosomal translocation [t(9;22)] in chronic myelogenous leukemia, is a substrate of PTPROt. Tyrosine phosphorylation of this potent kinase was markedly reduced in K562 cells expressing the catalytically active PTPROt. Enhanced dephosphorylation of Bcr-Abl by PTPROt both in vivo and in vitro explains the observed phenotypes of the PTPROt expressing K562 cells. These data taken together delineate the molecular mechanism of tumor suppressor function of PTPROt in leukemic cells characterized by Philadelphia chromosome. (This work was supported by a grant CA101956 from the National Institutes of Health).

High Throughput Methylation Arrays Allow for Identification of Complex Methylation Patterns in MDS.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2437-2437
Author(s):  
Ying Jiang ◽  
Christine L. OKeefe ◽  
Andrew Dunbar ◽  
Anjali Advani ◽  
Mikkael A. Sekeres ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
High Throughput ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Epigenetic Silencing ◽  
Low Grade ◽  
High Grade ◽  
Methylation Patterns ◽  
Hypermethylated Genes

Abstract Genomic imprinting and epigenetic silencing determine tissue-specific methylation patterns. Altered methylation of CpG islands within gene promoters has been hypothesized as one pathogenetic mechanism operative in myelodysplastic syndrome (MDS). Promoter hypermethylation of various empirically selected tumor suppressor genes has been found in MDS prompting application of hypomethylating drugs in this disease. Identification of hypermethylated genes predicting response to these drugs would have a major impact on clinical practice. However, to date methylation-based prognostic algorithms have not been established. Global analysis of DNA methylation patterns may help to identify hypermethylated genes/promoters associated with the pathogenesis of MDS. Recently, microarray-based DNA methylation analysis platforms enabled a powerful, high-throughput analysis of the methylation status of hundreds of genes. The GoldenGate Methylation Cancer Panel I, spanning 1,536 independent CpG sites selected from 807 selected genes was applied to determine the methylation status in MDS patients (N=51; 21 low grade (RA, MDS-U, RARS or RCMD), 26 high grade (AML or RAEB) and 4 CMML). The methylation status was determined based on an internal reference and compared to healthy controls (N=22). Methylation values were averaged among the patients or analyzed separately for each patient in comparison to average values obtained in controls. Overall, controls showed a lesser degree of methylation than advanced MDS patients (average intensity 0.326 vs. 0.339, p<0.05). Subsequently, we concentrated on hypermethylated genes. There were no genes uniformly hypermethylated in all patients. For 70%, 50%, and 30% of patients with advanced MDS, 1, 26, and 85 loci were concordantly hypermethylated, while in 70%, 50% and 30% of low risk patients 5, 23 and 31 were hypermethylated, respectively. The most consistently hypermethylated genes (>50% of patients), included tumor suppressor genes (DCC, SLC22A18, FAT, TUSC3), genes involved in DNA repair (OGG1, DDB2, BCR, PARP1), cell cycle control (DBC1, SMARCB1), differentiation (MYOD1, TDGF1, FGF2, NOTCH4) and apoptosis (HDAC1, ALOX12, AXIN1). Despite the variability, the aberrant methylation spectrum in CMML, low grade MDS and high grade MDS showed significant overlap (for example FZD9, IL16, EVI2A, MBD2 and BCR), which suggests that these genes may relate to the common tumorigenesis in MDS. Certain genes show specific methylation correlating to the morphologic diagnosis and may serve as diagnostic markers. For example, the promoter of HDAC1 is hypomethylated in 81% of sAML/RAEB1/2 patients but hypermethylated in 81% of low risk cases. To assess the link between epigenetic changes and chromosomal abnormalities, we also investigated methylation pattern of MDS with del5q for selected genes at the 5q locus. Some genes that are involved in apoptosis (WNT1, TNF receptor) and proliferation (MAP3K8, CSF3) were found to be hypermethylated in comparison to controls, suggesting that epigenetic silencing may enhance the effect of haploinsuffciency for some of the genes. In sum, our study, the first application of a high-throughput microarray methylation assay in MDS, demonstrates that complex methylation patterns exist in MDS and may allow for identification for clinically relevant methylation markers.

Up-Regulation of miR-195 Expression Leads to Decreased Expression of Basic Fibroblast Growth Factor in CLL Patients Treated with DNA Methylation Inhibitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3183-3183
Author(s):  
John D. Phillips ◽  
Ioana Pop ◽  
Ken Boucher ◽  
Margaret K. Yu
Keyword(s):  
Dna Methylation ◽  
Growth Factor ◽  
Tumor Suppressor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Dna Methyltransferase ◽  
Cpg Island ◽  
Fibroblast Growth ◽  
Substrate Inhibitor ◽  
Dna Methylation Inhibitors

Abstract A higher than age-expected DNA methylation index is predictive for early disease progression in patients with CLL (Yu et al. Leukemia Research 2006). We hypothesized that miRNA expression can also be silenced by promoter hypermethylation in CLL. Thus, methyl pool or DNA methyltransferase inhibitors can upregulate microRNAs with tumor suppressor characteristics by restoring the “normal” pattern of methylation. Results of patients treated with cladribine, a methyl substrate inhibitor, or 5-azacitidine, a DNA methyltransferase inhibitor were compared in 2 separate clinical protocols. The global DNA methylation decreased after treatment with cladribine or 5-azacitidine in 60% of the patients. At the 2006 ASH meeting, we reported on the consistent upregulation of miR-17-3p, miR-21, miR-29a, miR-29b, miR-29c, miR-30e, miR-104, miR-126, miR-128a, miR-130a, miR-141, miR-142-3p, miR-148a, miR-151, miR-199a, miR-199a*, and miR-301 by real-time PCR using the Early Access Human Panel from Applied Biosystems. Data from two patients on the cladribine protocol and 10 patients on the 5-azacitidine protocol were used for statistical analyses. Non-parametric Wilcoxon tests as well as t-tests were performed. Comparisons were made of the responders versus non-responders, and of cladribine versus 5-azacytidine. Since many of the miRNAs showed differences in the cladribine versus 5-azacytidine comparison, a second responder vs non-responder analysis was performed in which the two cladribine subjects were removed. MiR-195 was statistically more upregulated in the cladribine treated responder whereas miR-29c was statistically most upregulated in the 5-azacitidine treated patients (p=0.02). In the patients with global demethylation after treatment, upregulation of microRNA-195 was observed and directly correlated with regional demethylation of the CpG island, confirmed by bisulfite sequencing. Some of the predicted targets of miR-195 include bcl-2, CNOT6L, USP15, PADAH1B1, and ESRRG. MiR-195 may also have tumor suppressor characteristics as it also targets basic fibroblast growth factor (FGF-2), a gene important in CLL angiogenesis. There has been some evidence suggesting FGF-2 is an oncogene. For example, overexpression of FGF-2 isoforms facilitates growth of NIH 3T3 cells in low serum media and also mediates radioresistance of HeLa cells. FGF-2 is also protective against irradiation activation of p53 in the leukemia cells derived from patients with CLL. Although cladribine has been reported to downregulate FGF-2 by inhibiting adenosine deaminase, downregulation of FGF-2 at the transcript and protein levels was also observed in before and after treatment samples from patients treated with 5-azacytidine. We propose an alternative mechanism by which the FGF-2 transcript is degraded after binding to excess miR-195. In patients responsive to treatment with DNA methylation inhibitors, a regional decrease in the methylation status of the CpG island 5′ to miR-195 may lead to increased expression.

scholarly journals Cytopenia, Predisposition to Myelodysplastic Syndrome, Immunodeficiency, and Neurological Disease Caused By Gain-of-Function SAMD9L Mutations Is Frequently Ameliorated By Hematopoietic Revertant Mosaicism

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4299-4299
Author(s):  
Bianca Tesi ◽  
Josef Davidsson ◽  
Matthias Voss ◽  
Timothy Holmes ◽  
Tim Ripperger ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Myelodysplastic Syndrome ◽  
Nk Cell ◽  
Neutral Loss ◽  
Uniparental Disomy ◽  
Chromosome 7 ◽  
Hematopoietic Progenitor Cell ◽  
Gain Of Function ◽  
Monosomy 7 ◽  
Cytogenetic Aberrations

Abstract Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic diseases characterized by impaired hematopoiesis and progression to acute myeloid leukemia (AML). Although rare, several monogenic causes of familial MDS/AML have recently been molecularly defined. We studied two families with variable manifestation of cytopenia, MDS with cytogenetic aberrations involving chromosome 7, immunodeficiency, and neurologic disease consistent with ataxia-pancytopenia syndrome. Genetic studies uncovered heterozygous missense variants (p.Arg986Cys and p.Ile891Thr) in SAMD9L, a tumor suppressor gene located on chromosome 7. Consistent with a gain-of-function effect, transfection and over-expression of both SAMD9L variants decreased cell proliferation relative to wild-type protein. In the two families, a total of 10 individuals heterozygous for either SAMD9L mutation were identified. Three individuals developed MDS, with monosomy 7 or der(1;7)(q10;p10) as cytogenetic aberrations that encompassed the mutant SAMD9L locus. In an additional five individuals, three of which experienced a spontaneously resolved cytopenic episodes in infancy, we detected mosaic copy-neutral loss of heterozygosity of 7q by uniparental disomy, with loss of the mutated allele, or mosaic cis SAMD9L mutations. By digital PCR, we identified these events in hematopoietic progenitor cell populations, which were further enriched in B and NK cell lineages. Absent in non hematological tissues, these mutations thus represented somatic revertant mosaicism. Clinically, revertant mosaicism was associated with reduced disease severity, although in two individuals neurological manifestations persisted. Of note, two unaffected carriers without revertant mosaicism harbored an additional rare in trans germline SAMD9L p.Thr233Asn missense variant. In cellular assays, the SAMD9L p.Thr233Asn variant increased proliferation, indicating a loss-of-function effect that potentially compensates for the SAMD9L p.Arg986Cys mutation. Together, our results demonstrate that gain-of-function mutations in the tumor suppressor SAMD9L cause a disease with cytopenia, immunodeficiency, variable neurological presentation, and predisposition to MDS with chromosome 7 aberrations, where hematopoietic revertant mosaicism is common and ameliorates the clinical presentation. Disclosures Fioretos: Cantargia: Equity Ownership.

High-Resolution Genomic Arrays Facilitate Detection of Novel Cryptic Chromosomal Lesions in MDS.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 370-370
Author(s):  
Christine L. O’Keefe ◽  
Ramon Tiu ◽  
Lukasz Gondek ◽  
Aaron Viny ◽  
Karl Theil ◽  
...  
Keyword(s):  
High Resolution ◽  
Chromosomal Abnormalities ◽  
Bone Marrow Cells ◽  
Genomic Analysis ◽  
Normal Karyotype ◽  
Chromosome 7 ◽  
Comparative Genomic ◽  
Stem Loop ◽  
Monosomy 7 ◽  
Normal Cytogenetics

Abstract The evolution of abnormal hematopoietic clones characterized by acquired chromosomal abnormalities is the central event in the pathogenesis of MDS. Defective chromosomes have significant clinical implications in the management of MDS and suggest the presence of an inherent chromosomal instability. As karyotypic lesions are not found in all MDS patients, it is possible that in some the dysplastic clone may evolve without a chromosomal defect or, more likely, the resolution of routine metaphase cytogenetics is not sufficient to detect smaller lesions; in many instances lack of growth precludes the analysis. Array-based comparative genomic hybridization (A-CGH) allows for a high-resolution genomic scan that circumvents some of the limitations associated with the use of conventional cytogenetics. We hypothesized that high-resolution genomic analysis of genetic gains and losses by A-CGH may detect cryptic lesions, particularly in patients with negative/non-informative cytogenetics that may be of clinical/scientific significance. We examined bone marrow cells from 39 MDS patients (18 RA/RARS, 11 RAEB-t, 6 CMML and 4 secondary AML) and 11 controls using a 2632 BAC microarray and CGH. Dye swapping on duplicate arrays assured reproducibility of the CGH results, confirmed globally by a high resolution 50K SNP microarray in 4 patients and by microsatellite analysis in others. By traditional cytogenetics 19 patients had chromosomal lesions, 18 were normal and 2 tests non-informative. When A-CGH was applied, a normal karyotype was found in only 15% of patients in comparison to 46% by metaphase cytogenetics. Of note is that both cases with uninformative cytogenetics showed an abnormal CGH result and in several patients (N=11) with an abnormal karyotype additional lesions were found. Karyotypic results were confirmed in 7 cases; discordant analysis may be due to a lower proportion of dysplastic cells in marrow. Irrespective of the genomic area affected, when we studied the raw number of lesions more advanced forms of MDS (RAEB-t/AML) were evenly distributed between patients subdivided on sheer number of lesions (0, 1–17, >17). Many hotspots of genomic instability shared between patients were identified. For example, 1p26.3, 10q26 and 4p16 lesions were found in 2 or more patients. Interestingly, these regions contain genes of potential pathologic significance, including tubulin gamma complex associated protein 2 (TUBGCR2) and histone stem-loop binding protein (SLBP). Cryptic lesions on chromosome 7 (e.g. 7p21, 7q31) were identified in 5 patients with normal cytogenetics. These patients suffered from severe cytopenias, consistent with the prognosis of monosomy 7 and highlighting a consensus defect on chromosome 7. Certain chromosomes were rarely or never affected, implying that a more targeted array might be designed for clinical use. A-CGH Cytogenetics Unsuccessful Normal Abnormal Unsuccessful (N=2) 0 0 2 Normal (N=18) 0 3 15 Abnormal (n=19) 0 3 16 In summary, our study highlights the superior level of resolution of A-CGH as compared to metaphase analysis in the diagnosis of MDS. A prospective analysis is underway to determine the prognostic value of CGH-detected lesions and their pathophysiologic significance.

Abnormal Mitosis and Genetic Instability Induced by Downregulation of Candidate Myeloid Tumor Suppressor Genes Isolated from the Long Arm of Chromosome 7.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 390-390
Author(s):  
Hiroya Asou ◽  
Hirotaka Matsui ◽  
Yuko Ozaki ◽  
Toshiyuki Takemura ◽  
Akiko Nagamachi ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Genetic Instability ◽  
Tumor Suppressor Genes ◽  
Leukemia Cell ◽  
Chromosome 7 ◽  
Gene Products ◽  
Suppressor Genes ◽  
Monosomy 7 ◽  
Ancestral Gene

Abstract To isolate myeloid tumor suppressor genes from 7q, we tried to detect microdeletions (< 100 Kb) that might be present in MDS/AML cells carrying apparently normal chromosome 7. For this purpose, we developed our original microarray-based CGH technology. In this system, instead of BAC clones generally used as probes, we applied short (3–5 Kb) genomic DNA fragments containing strictly no repetitive sequences. We made 235 probes in a region spanning 21.7 Mb within 7q21.3–7q31.1. Although we selected MDS/AML patients whose marrow did not show cytogenetically visible 7q deletions, gross copy number changes frequently observed in adult patients prevented us from identification of common microdeletions. By investigation of 21 childhood myeloid leukemia patients with normal karyotype, we successfully identified a common microdeletion spanning approximately 120 Kb. Eight (38%) patients shared this microdeletion, which was not detected in normal individuals. Database search revealed that this region contains three hypothetical genes. Only vertebrates have these genes that likely evolved from one common ancestral gene of fish. Real-time quantitative PCR revealed that 9 (29%) out of 31 adult MDS/AML harbors microdeletions in at least one of these three genes. None of these genes had been well characterized nor has known motifs that would suggest function of the gene products. We named them Miki, Titan and Kasumi. Immunoblot analysis revealed expression of all three genes at high levels in most lymphoid leukemia cell lines, while half of myeloid cell lines lacked at least one of their expression. In leukemia cells carrying monosomy 7, expression levels were generally low. Miki, a heavily glycosylated protein, co-localized with centrosomes and spindles in the mitotic phase. To test the function of Miki, we used si-RNAs to downregulate Miki expression in HeLa and K562 cells, both of which show basically normal metaphase and nuclear morphology. Cells expressing Miki at reduced levels showed small and fragmented centrosomes, loss of spindle tension, tripolar mitosis or even completely disturbed spindle formation. As a result, anaphase lagging, colchicine-mitosis (C-mitosis), premature chromosome decondensation and chromatid bridges were observed in virtually all cells in the mitotic phase. In the interphase, bi- or tri-nuclear or even multinuclear cells with micronuclei, all of which are characteristic to MDS, were frequently observed. On the other hand, proteomic analysis revealed that Titan and Kasumi bind to the DNA-PK complex, which plays critical roles in the non-homologous end joining (NHEJ) of double stranded DNA (dsDNA) breaks. Indeed, these proteins were translocated from cytoplasm to nucleus by ionizing radiation (IR) or by treatment of drugs that yield dsDNA breaks. Cells expressing Kasumi at reduced levels by si-RNA showed increased radiosensitivity, sister chromatid exchange, and number of background-level phosphorylated histone H2AX foci (i.e., foci formed without IR), which are co-localized with dsDNA breaks. These results indicate that the genes we isolated are promising candidates for anti-leukemic genes located in 7q, because downregulation of these gene products by 7q deletions would cause the abnormal morphology of MDS and genetic instability.

Eradication and Reduction of the Abnormal Chromosome 7 Clone with Azacitidine (AZA C) Is Achievable for One-Third of Patients (pts) with Myelodysplastic Syndrome (MDS).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5181-5181
Author(s):  
Vesna Najfeld ◽  
Angela Scalise ◽  
Rosalie Odchimar-Reissig ◽  
Lewis R. Silverman
Keyword(s):  
Longitudinal Study ◽  
Stable Disease ◽  
Normal Karyotype ◽  
Cytogenetic Response ◽  
Abnormal Chromosome ◽  
Chromosome 7 ◽  
Abnormal Karyotype ◽  
Monosomy 7 ◽  
Cytogenetic Abnormalities ◽  
Results Of Treatment

Abstract The two most frequent cytogenetic abnormalities in pts with MDS involve rearrangements of chromosomes 5 and 7. Monosomy 7 or deletion 7q, alone or in a complex karyotypes are poor-risk abnormalities and associated with a low response rate to conventional therapies. As a part of our comprehensive longitudinal study of 220 patients treated with AZA C, we asked the question whether pts with −7/del(7q) alone or as a part of the complex clone, may achieve hematological and/or cytogenetic response. A minimum of three follow-up cytogenetic and FISH analyses were required as an inclusion criterion. A normal karyotype was observed in 129 of 229 pts (56%) and 100 pts (44%) had an abnormal karyotype at baseline. Among the 100 pts with an abnormal karyotype 29 pts (29%) had chromosome 7 abnormalities prior to the AZA C treatment. In an additional 12 pts chromosome 7 abnormalities developed during the course of disease and AZA C therapy (range 4 months to 5 years). Response of the abnormal chromosome 7 clone to AZA C therapy was observed in 3 patterns: pts who had −7/del(7q) present at baseline without any further cytogenetic change during the AZA C treatment (20 pts; 69%). Eight of these pts had hematological improvement (HI); Pts who had either reduction or elimination (complete cytogenetic response=CCR) of the abnormal chromosome 7 clone as a results of treatment (9 pts, 31%). Four of 29 pts (14%) had a reduction in the size of the abnormal clone from 100% to a mean of 23% (range 8%–50%) as judged by conventional cytogenetics. The median time to achieve reduction was 6 months (range 2 to 9 months) with median duration of 10.5 months (range 6 to 24 months) during maintenance therapy with AZA C. CCR was observed in 5 of 29 pts (17%) and occurred within a mean of 4.2 months (range 3–6 months). Repeated cytogenetic studies showed a normal karyotype and the CCR lasted a mean of 5.2 months (range 3–9 months) during therapy. FISH studies showed 2–5% cells with −7/del(7q) during the CCR. Four of the 5 pts had HI and one pt had CR. These patients relapsed with the diagnostic −7/del(7q) clone without additional cytogenetic abnormalities. 3) Of the 12 pts who developed −7/del(7q) while on Aza C therapy, 4 pts had a normal karyotype at baseline and developed −7/del(7q) after a mean of 16 months (range 6–39 months) therapy. One had PR and 3 pts had a stable disease. The other 8 pts were cytogenetically abnormal at baseline and developed −7/del(7q) as a subclonal evolution during therapy in 4 pts (range 4 months to 5 years) and following disease progression in 4 pts. Six pts had HI and two had a stable disease. In conclusion, our longitudinal study allowed us to delineate 3 categories of AZA C response to abnormal chromosome 7 clone: 31% had cytogenetic response: 17% complete and 14% major and 69% had no change in the abnormal −7/del(7q) clone. In additional 5% (12/229) pts, −7/del(7q) emerged either as a new abnormal clone or as a subclonal evolution during therapy or at progression. AZA C-based therapy can either stabilize or reduce/eradicate the abnormal chromosome 7 clone as determined by both, cytogenetics and FISH.

IKZF1 deletions Are Detected In 40.3% Of 270 Adult B-Precursor-ALL and Are Independently Associated With BCR-ABL1 Positivity and Inferior Outcome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1372-1372
Author(s):  
Annette Fasan ◽  
Claudia Haferlach ◽  
Madlen Ulke ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Normal Karyotype ◽  
Chromosome 7 ◽  
Employment Equity ◽  
Gene Deletions ◽  
Exon 2 ◽  
Monosomy 7 ◽  
Significant Difference ◽  
Equity Ownership ◽  
Exon 4

Abstract Introduction Total or intragenic deletions of or within the IKZF1 (Ikaros) gene on chromosome 7 have been shown to be frequent in B-precursor-ALL (B-ALL), highly associated with BCR-ABL1 positive (BCR-ABL1pos) B-ALL (Mullighan et al., Nature 2008), but also present in BCR-ABL1 negative (BCR-ABL1neg) B-ALL (Mullighan et al., N Engl J Med 2009). IKZF1 deletion pattern is known to be heterogenous and deletions involving exon 4, 7 or 8 are likely to have the same impact as whole gene deletions. To analyse intragenic IKZF1 deletions, we used a multiplex-PCR approach, which represents the standard detection method. In addition, we used multiplex ligation-dependent probe amplification (MLPA), which allows the detection of whole IKZF1 deletions. Aim We aimed at characterization of IKZF1 deletions pattern and correlation to clinical features in a cohort of 270 adult B-ALL cases. Patients and Methods IKZF1 deletion status was analyzed in blood or bone marrow samples from 270 adult B-ALL cases (subtypes as diagnosed by immunophenotyping: c-ALL: n=197; Pro-B ALL: n=51; Pre-B ALL: n=5). Additionally, 17 mature B-ALL cases were analyzed. The cohort consisted of 137 females and 133 males, median age was 58.3 years (range: 18.1-91.4 years). The cohort was classified into seven subgroups according to the following cytogenetics: 1) t(9;22)(q34;q11) (n=97), 2) 11q23/MLL rearrangements (n=24), 3) MYC rearrangements (n=14), 4) hypodiploidy (n=21), 5) hyperdiploidy (n=32), 6) normal karyotype (n=38), 7) other cytogenetic aberrations (n=42). In one case no cytogenetic data was available. In all 270 cases intragenic IKZF1 deletions were investigated by breakpoint-specific fluorescent multiplex PCR (according to Caye et al., Hematologica 2012). In 206 cases we additionally used MLPA (P335 SALSA MLPA kit IKZF1, MCR Holland, The Netherlands) to identify whole IKZF1 deletions not detectable by multiplex-PCR. Results In total, 132 IKZF1 alterations were identified in 109/270 cases (40.3%). With regard to ALL subtypes in c-ALL 47.7% (94/197) IKZF1 mutations were detected, in Pro-B ALL 23.5% (12/51) and in three of five Pre-B ALL cases. No IKZF1 deletions were detected in mature B-ALL and thus were mutually exclusive with MYC rearrangements. There was no significant difference in age, sex, leukocyte count, hemoglobin level or platelet count between patients with or without IKZF1 deletions, respectively. With regard to immunophenotype cases with IKZF1 deletions had a stronger expression of CD13 (38±28% vs. 27±25% positive cells; p=0.004), CD33 (24±23% vs. 17±21%; p=0.008), CD34 (72±25% vs. 43±35%; p<0.001%) and TdT (61±28% vs. 43±32%; p<0.001). In 86/109 cases (78.9%) one (monoallelic) intragenic IKZF1 deletion was detected by multiplex-PCR and MLPA. In detail, 38 patients (34.9%) showed deletions of exon 4-7, 15 cases (13.7%) deletions of exon 2-7, 5 cases (4.6%) deletions of exon 4-8 and 3 cases (1.8%) deletions of exon 2-8. In one case, a deletion of IKZF1 exons 2 and 3 was detected by MLPA. 23/109 cases (21.1%) showed two (biallelic) IKZF1 deletions. Whole IKZF1 gene deletions were detected by MLPA in 24/109 cases (22.0%) and were associated with cytogenetic abnormalities of the short arm of chromosome 7 in 21/40 cases (52.5%) including: monosomy 7 (n=14), i(7)(q10) (n=2), dicentric chromosomes 7 (n=1), and unbalanced translocations involving chromosome 7 (n=4). Four of 24 cases with MLL-rearrangements harbored IKZF1 deletions, three of these being whole IKZF1 gene deletions due to monosomy 7. IKZF1 deletions were highly associated with BCR-ABL1 positivity: 72/97 BCR-ABL1pos (74.2%) versus 37/172 BCR-ABL1neg cases (21.5%) (p<0.001). 61/85 cases (71.8%) with intragenic IKZF1 deletions were BCR-ABL1pos compared to 11/24 (45.8%) cases with whole IKZF1 deletions (p=0.018). In 97 BCR-ABL1pos cases, the additional presence of IKZF1 deletions was correlated to inferior survival (p=0.070). Conclusions 1) IKZF1 deletions were detected in 40.3% in adult B-ALL by using muliplex-PCR and MLPA. 2) Whole but also intragenic IKZF1 deletions are highly associated with BCR-ABL1 in adult B-ALL. 3) As 22% of IKZF1 deletions were whole gene deletions not detectable by standard multiplex-PCR, at least two molecular methods (multiplex-PCR and MLPA) are required to fully detect the whole spectrum of IKZF1 deletion patterns in adult B-ALL. Disclosures: Fasan: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ulke:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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