Wilms Tumor 1 Gene Mutations in Adult Acute T-Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2516-2516
Author(s):  
Sandra Heesch ◽  
Nicola Goekbuget ◽  
Jutta Ortiz Tanchez ◽  
Cornelia Schlee ◽  
Stefan Schwartz ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Wilms Tumor ◽  
Lymphoblastic Leukemia ◽  
Single Amino Acid ◽  
Newly Diagnosed ◽  
Expression Levels ◽  
Wilms Tumor 1 ◽  
Standard Risk ◽  
Wt1 Protein ◽  
Mrna Expression Levels

Abstract The wilms tumor 1 gene (WT1) encodes a transcriptional regulator involved in normal hematopoietic development. The role of WT1 in acute leukemia has been underscored by the finding of WT1 overexpression in subsets of patients (pts) associated with an increased relapse risk. In addition mutations of WT1 have been found in about 10–15% of acute myeloid leukemia (AML) pts and have recently shown to predict inferior survival. Thus far, larger studies have not yet determined the frequency and impact of WT1 mutations in acute T-lymphoblastic leukemia (T-ALL). Herein, we have analyzed WT1 mutations and WT1 mRNA expression levels in a large cohort of T-ALL including 239 newly diagnosed adult pts treated on the GMALL protocols 0699 and 0703. Diagnostic bone marrow specimens were studied for WT1 mutations by DNA sequencing. In addition, samples were immunophenotyped, and mRNA expression of the molecular markers HOX11, HOX11L2, ERG, BAALC, as well as WT1 were determined by real-time RT-PCR. Twenty (8%) of the 239 analyzed T-ALL pts had WT1 mutations (WT1mut) [20 pts had mutations in exon 7 (WT1mut7), with 2 pts having coexisting mutations in exon 9 (WT1mut9)]. WT1mut7 were frameshift or nonsense mutations predicted to result in a truncated WT1 protein, whereas WT1mut9 were missense mutations leading to single amino-acid substitutions. WT1mut and WT1 wildtype (WTwt) pts did not significantly differ with respect to clinical parameters at diagnosis (e. g. age, leukocyte count, and sex). WT1mut cases were characterized by immature features such as an early immunophenotype (45% of WT1mut showed an early T-ALL immunophenotype as compared to only 25% of WT1wt), and WT1mut also showed higher levels of CD34 expression as determined by flow cytometry (WT1mut median: 46% vs. WT1wt median: 2 %; P=0.03). Moreover, WT1mut had significantly higher WT1 mRNA expression levels [WT1mut median: 0.05 (range: 0–0.395) vs. WT1wt median: 0 (range: 0–0.15); P<0.001]. Significant differences were not observed in the complete remission rate nor overall survival or relapse free-survival (RFS) between WT1mut and WT1wt pts. However, in the standard risk group of thymic T-ALL 80% (4/5) of WT1mut relapsed as compared to 28% (25/89) of WT1wt thymic pts [P=0.01; RFS at 18 months: 20% (SE: ±18) for thymic WT1mut vs. 82% (SE: ±4) for thymic WT1wt pts; P=0.008]. In conclusion, in adult T-ALL WT1 mutations are present in 8% of newly diagnosed pts and are located in the same region as reported in AML expected to impair the DNA binding ability of the WT1 protein. Similar to findings in AML, WT1mut cases are characterized by immature features pinpointing to a genetic hit in hematopoietic progenitors likely harboring bilineage potential. The prognostic implications of WT1 mutations in standard risk thymic T-ALL will have to be further validated in independent studies and may in future direct molecularly-based treatment stratification.

scholarly journals Prognostic significance of Wilms tumor 1 mRNA expression levels in peripheral blood and bone marrow in patients with myelodysplastic syndromes

2016 ◽  
Vol 17 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Sumiko Kobayashi ◽  
Yasunori Ueda ◽  
Yasuhito Nannya ◽  
Hirohiko Shibayama ◽  
Hideto Tamura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mrna Expression ◽  
Myelodysplastic Syndromes ◽  
Peripheral Blood ◽  
Wilms Tumor ◽  
Prognostic Significance ◽  
Expression Levels ◽  
Wilms Tumor 1 ◽  
Mrna Expression Levels

Diversity of Class I Major Histocompatibility Genes From Divergent Haplotypes in Zebrafish

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1058-1058
Author(s):  
Sean C. McConnell ◽  
Jill L. O. de Jong
Keyword(s):  
Mrna Expression ◽  
Class I ◽  
Single Amino Acid ◽  
Gene Copy ◽  
Major Histocompatibility ◽  
Chromosome 19 ◽  
Expression Levels ◽  
Transplantation Experiments ◽  
Haplotype A ◽  
Mrna Expression Levels

Abstract Abstract 1058 The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines, particularly in the context of transplantation experiments that distinguish between autologous and allogeneic tissues. Major histocompatibility (MH) genes are considered the most polymorphic genes in vertebrates, yet this immense variation occurs while maintaining conserved roles in antigen presentation. Histocompatibility in zebrafish remains poorly understood, requiring the identification of the classical MH genes as well as an evaluation of the variation between haplotypes. Although at least 11 putative zebrafish Class I MH U lineage genes have been isolated from cDNA libraries, their genomic organization and haplotype assignments remain uncharacterized for the majority. We focused our study on a set of diverse zebrafish Class I MH genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching was previously shown to be associated with improved engraftment after transplantation. Inside the conserved psmb8 and tpsn flanking gene regions on chromosome 19, Class I MH haplotypes can differ markedly among zebrafish strains including Tubingen and AB. Interestingly, the distinct haplotypes at chromosome 19 appear to maintain non-overlapping sets of genes, and also have gene copy number differences. For example, haplotype A contains the genes mhc1uda, mhc1uea, and mhc1ufa, with tap2 genes located in between each set of MH genes. In contrast, haplotype B contains mhc1uba and mhc1uca genes that are separated instead by a tapbp gene. Among these two highly divergent haplotypes, mhc1uea and mhc1uda from haplotype A appear to be more closely related to one another than to either of the MH genes on haplotype B. Similarly, the mhc1uba and mhc1uca genes from haplotype B share higher levels of sequence identity than in comparison with genes from haplotype A. These findings differ markedly from the MH genes in mammals where the highest similarity is between alleles of the same Class I gene (for example, HLA-A in humans), instead of the other genes on the same haplotype (eg. HLA-B and HLA-C). In addition, the zebrafish MH gene sequences are very closely related to MH genes from other fish species. As an example, mhc1ufa is more similar to a Class I MH gene from gibuna carp than to other zebrafish MH genes. These data in zebrafish indicate that selected Class I MH gene polymorphisms have been preserved within the species since before the species diverged from other teleost fishes, suggesting a selective advantage for this unique form of MH diversity. To determine mRNA expression levels of the Class I MH genes, quantitative PCR was performed on liver, spleen, kidney marrow, testis, gill, intestine and heart from zebrafish that were homozygous for either haplotype A or haplotype B. Within haplotype A, expression of mhc1uda, mhc1uea and mhc1ufa is relatively equivalent, with mhc1ufa expressed at the highest levels overall. For haplotype B, mhc1uba has the highest levels in all tissues examined, while mhc1uca expression is 10–100 fold lower and somewhat variable between tissues. Putative peptide anchor residues are highly conserved between species. Those anchor residues that are conserved in all species including mammals, as well as two additional residues that are conserved in teleosts, are also all conserved in mhc1uda, mhc1uba, and mhc1uca. In contrast, mhc1uea and mhce1ufa each have single amino acid substitutions at critical residues, K146N and Y59F, respectively. These substitutions do not necessarily disqualify these molecules from consideration as classical MH Class I genes. In conclusion, we propose that mhc1uba and mhc1uda function as classical MH Class I genes within divergent haplotypes, based on mRNA expression levels and tissue distribution, as well as sequence properties including conservation of putative peptide anchor residues. Using these same criteria, additional zebrafish Class I MH genes may be identified to serve similar roles. These predictions require verification via additional functional transplantation experiments that are currently underway in our lab. Defining the functional zebrafish Class I MH genes will provide an important foundation for future studies in immunology and transplantation. Disclosures: No relevant conflicts of interest to declare.

2′-Deoxy-5-Azacytidine Enhances Cytotoxic Effect Of Other Anti-Leukemic Agents Synergistically In Acute Lymphoblastic Leukemia Cell Line

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5564-5564
Author(s):  
Kimiyoshi Sakaguchi ◽  
Hiroyoshi Takahashi
Keyword(s):  
Mrna Expression ◽  
Mtt Assay ◽  
Research Funding ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Expression Levels ◽  
Apoptotic Genes ◽  
Mrna Expression Levels

Abstract Introduction Advances in chemotherapy have improved the outcome of childhood acute lymphoblastic leukemia (ALL). However, leukemia cells in refractory ALL are often resistant to anti-leukemic agents. Although recent studies have focused on the epigenetic changes in refractory leukemia, the relationship between the demethylating agent 2′-deoxy-5-azacytidine (decitabine, DAC) and ALL remains unclear. Here, we examine the combined effects of DAC and anti-leukemic agents such as clofarabine (CLO) and etoposide (ETO) on the ALL cell line CCRF-CEM. Methods and results In vitro drug sensitivity was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. We cultured CCRF-CEM cells for 72 hours with or without DAC, and then removed DAC (when present) prior to culturing CCRF-CEM cells for 48 hours with ETO or CLO, or without chemotherapeutic drugs. After culturing for 48 hours, we removed the chemotherapeutic drugs and measured in vitro drug sensitivity using MTT assay. The MTT assay was performed in triplicate. We then evaluated the inhibitory concentration at 50% (IC50). IC50 for ETO, ETO+DAC, CLO, and CLO+DAC was 3.36, 0.625, 4.96, and 1.92, respectively. The combination Index (CI) was produced with Calcusyn® software, which uses the methodology of Chou and Talalay to perform formal synergy analyses. A CI < 1 indicated a synergistic effect. The CI was 0.026 for ETO+DAC and 0.431 for CLO+DAC. We assayed with Annexin-V, PI staining, and caspase-3/7 to detect apoptosis. We observed apoptosis rates of 31.6%, 53.3%, 31.2%, and 52.6% for ETO, ETO+DAC, CLO, and CLO+DAC, respectively. We observed greater caspase-3/7 activity with DAC+CLO and DAC+ETO than with CLO and ETO. Using real-time reverse transcription polymerase chain reaction (RT-qPCR) in CCRF-CEM cells, we examined mRNA expression levels for the pro-apoptotic genes BAK, BID, BAX, BAD, BIM, PUMA, ATM, TP53, and NOXA, as well as those for the anti-apoptotic genes BCL2, BCL2L1, and XIAP. The expression level of each target gene was calculated by normalizing it to the housekeeping gene GAPDH. The RT-qPCR was performed in triplicate. We used Student’s t test to compare the data. We observed DAC increased mRNA expression levels of BAX and NOXA, but decreased those for BAK, BID, PUMA, BCL2L1, ATM, TP53, and XIAP. We then analyzed the methylation status of pro- and anti-apoptotic genes after 48 hours incubation with or without DAC. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation with DAC was 1.3%, 3.3%, 2.5% and 72.9%, respectively. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation without DAC was 1.9%, 3.6%, 0.7% and 92.3%, respectively. There was no significant difference. Discussion Our results showed that DAC synergistically enhances CLO and ETO cytotoxicity, and this cytotoxic effect depends on caspase-3/7 activity. We examined mRNA expression levels of pro- and anti-apoptotic genes. We hypothesized that DAC would increase mRNA expression levels of most pro-apoptotic genes, and decrease mRNA levels of most anti-apoptotic genes. We found that DAC decreased some pro-apoptotic genes, such as BAK, BID, PUMA, ATM, and TP53, which disproves our hypothesis. Our present findings are similar to those of Shin et al., who reported that DAC decreased BID mRNA expression levels. However, they provided no explanation for this activity. Our results show that DAC did not demethylate the CpG of BAK, NOXA, BCL2L1, or XIAP. Thus, DAC must demethylate the CpG of other genes. Nevertheless, many genes are involved in apoptosis, and it remains unclear which genes are demethylated by DAC. Disclosures: Sakaguchi: Yakult Honsha Company: Research Funding; Japan Leukemia Research Fund: Research Funding; Japan Society for the Promotion of Science: Research Funding; Sanofi: Research Funding; Teijin Pharma: Research Funding.

RPL5 Is a Candidate Tumor Suppressor on 1p22.1 in Multiple Myeloma of Which the Expression Is Linked to Bortezomib Response

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2969-2969
Author(s):  
Isabel JF Hofman ◽  
Mark van Duin ◽  
George Mulligan ◽  
Ellen Geerdens ◽  
Emanuela Garelli ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Suppressor ◽  
Mrna Expression ◽  
Integration Site ◽  
Lymphoblastic Leukemia ◽  
Expression Level ◽  
Phase Iii ◽  
Newly Diagnosed ◽  
Expression Levels

Abstract Chromosomal region 1p22 is deleted in ~20% of multiple myeloma patients, suggesting the presence of an unidentified tumor suppressor gene in this region. Using high resolution copy number arrays, we delimit a 58 kb minimal deleted region on 1p22.1 encompassing two genes: ectopic viral integration site 5 (EVI5) and ribosomal protein L5 (RPL5). Although mutations in 1p22 genes are rare in multiple myeloma, the tumor suppressor role of EVI5 and RPL5 may be supported by the fact that these genes show the highest frequency of mutations predicted to impair protein function on 1p22. Interestingly, inactivation of RPL5 was also recently described in T-cell acute lymphoblastic leukemia (T-ALL) and glioblastoma. We found that 1p22 deleted patients have significantly lower levels of EVI5 and RPL5 mRNA expression (59% and 71% residual expression respectively; p<0.0001 for each gene). Whereas 1p22 deletion status correlates well with EVI5 expression in all patients, it is a bad predictor of RPL5 expression level in some cases, suggesting that other mechanisms besides 1p22 deletion can downregulate RPL5 expression in multiple myeloma. Deletion of 1p22 has been associated with decreased progression free (PFS) and overall survival (OS) in newly diagnosed multiple myeloma (Hebraud et al., Leukemia, 2013). We saw that low mRNA expression of EVI5 and RPL5 also correlates with worse survival in diagnosis but not in relapse cases. Interestingly, RPL5 but not EVI5 mRNA expression levels were significantly lower in multiple myeloma patients responding to bortezomib compared to patients that are not responding (68% residual RPL5 expression in responders versus non-responders). The clinical relevance of these findings is illustrated by the observation that PFS of newly diagnosed patients with low RPL5 expression was significantly longer when they were treated on a bortezomib containing protocol (median PFS bortezomib protocol 29.8 months versus 18.7 months for non-bortezomib, p=0.03, data phase III HOVON-65/ GMMG-HD4 trial). In contrast, PFS of RPL5 high patients was not influenced by bortezomib. These associations between RPL5 expression level and bortezomib response were confirmed when performing the same analyses on the PFS data from relapse patients in the APEX trial. For low EVI5 expressing patients, there was a non-significant trend towards benefit from bortezomib which was not present in the EVI5 high cases. Taken together, these data suggest that RPL5 expression levels are a better biomarker for bortezomib response than 1p22 deletion. In conclusion, we provide genetic data that narrow down the list of candidate tumor suppressors on 1p22 to EVI5 and RPL5. Although the exact role of these genes in suppressing multiple myeloma disease progression remains to be determined, we identify low RPL5 expression levels in multiple myeloma as a novel, clinically relevant biomarker associated with initial response and survival benefit upon treatment with bortezomib. Disclosures Mulligan: Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Delforge:Amgen: Honoraria; Novartis: Honoraria; Celgene Corporation: Honoraria; Janssen: Honoraria. Sonneveld:Janssen-Cilag, Celgene, Onyx, Karyopharm: Honoraria, Research Funding; novartis: Honoraria.

scholarly journals The role of CXCR3 and its ligands expression in Brucellar spondylitis

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Xin Hu ◽  
Xiaoqian Shang ◽  
Liang Wang ◽  
Jiahui Fan ◽  
Yue Wang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Inflammatory Infiltration ◽  
Peripheral Blood Mononuclear ◽  
Expression Levels ◽  
Inflammatory Damage ◽  
Ifn Γ ◽  
Mrna Expression Levels

Abstract Aim Brucellar spondylitis (BS) is one of the most serious complications of brucellosis. CXCR3 is closely related to the severity of disease infection. This research aimed to study the degree of BS inflammatory damage through analyzing the expression levels of CXCR3 and its ligands (CXCL9 and CXCL10) in patients with BS. Methods A total of 29 BS patients and 15 healthy controls were enrolled. Real-Time PCR was used to detect the mRNA expression levels of IFN-γ, CXCR3, CXCL9 and CXCL10 in peripheral blood mononuclear cells (PBMCs) of BS patients and healthy controls. Hematoxylin-Eosin staining was used to show the pathological changes in BS lesion tissues. Immunohistochemistry staining was used to show the protein expression levels of Brucella-Ab, IFN-γ, CXCR3, CXCL9 and CXCL10 in BS lesion tissues. At the same time, ELISA was used to detect the serum levels of IFN-γ, CXCL9 CXCL10 and autoantibodies against CXCR3 in patients with BS. Results In lesion tissue of BS patients, it showed necrosis of cartilage, acute or chronic inflammatory infiltration. Brucella-Ab protein was abundantly expressed in close lesion tissue. And the protein expression levels of IFN-γ, CXCR3 and CXCL10 were highly expressed in close lesion tissue and serum of BS patients. At the same time, the mRNA expression levels of IFN-γ, CXCR3 and CXCL10 in PBMCs of BS patients were significantly higher than those in controls. Conclusion In our research, the expression levels of IFN-γ, CXCR3 and its ligands were significantly higher than those in controls. It suggested that high expression levels of IFN-γ, CXCR3 and its ligands indicated a serious inflammatory damage in patients with BS.

Unilateral labyrinthectomy induced changes on GDNF receptor complex proteins in the rat medial vestibular nuclei

2007 ◽  
Vol 16 (4-5) ◽  
pp. 171-177
Author(s):  
Adrian Lozada ◽  
Kaj Karlstedt ◽  
Pertti Panula ◽  
Antti A. Aarnisalo
Keyword(s):  
Mrna Expression ◽  
Vestibular Nucleus ◽  
Medial Vestibular Nucleus ◽  
Receptor Complex ◽  
Trophic Effect ◽  
Expression Levels ◽  
Protein Levels ◽  
Unilateral Labyrinthectomy ◽  
Ganglion Neurons ◽  
Mrna Expression Levels

In the auditory periphery, GDNF has been shown to have a trophic effect to spiral ganglion neurons, both during development and in adult animals. We have studied the effect of unilateral labyrinthectomy (UL) on protein levels and expression of GDNF multicomponent receptor complex: the ret tyrosine kinase and coreceptor GFRα-1 in the medial vestibular nucleus of the adult rat. GFRα-1 protein levels display an increasing trend in ipsilateral medial vestibular nucleus culminating at 48 h post UL. On the other hand, GFRα-1 mRNA expression levels in ipsi- and contralateral medial vestibular nucleus show a steadily decreasing trend that is significant at 1 week post-lesion. Protein levels for c-Ret isoforms also show an initial bilateral decreasing trend that ceases at 48 h in ipsilateral medial vestibular nucleus but persists on the contralateral side. c-Ret mRNA expression levels show a significant decrease at 4 h post UL followed by another significant decrease 1 week post UL. Our data would suggest that neurotrophins belonging to the GDNF family are involved in this model of post-lesional CNS plasticity.

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