The Experts below are selected from a list of 4716 Experts worldwide ranked by ideXlab platform
David Oscier - One of the best experts on this subject based on the ideXlab platform.
-
ZAP70 Methylation Is An Independent Prognostic Biomarker For Front Line Therapy Of Chronic Lymphocytic Leukemia : Results From The UK LRF CLL4 Trial
Blood, 2013Co-Authors: Ben Gregory, Zadie Davis, Monica Else, Matthew Jj Rose-zerilli, Jonathan C. Strefford, Daniel Catovsky, David OscierAbstract:In CLL, over-expression of ZAP70 is well established as a prognostic biomarker for unfavourable disease course. However, technical challenges limit the application of flow cytometry for ZAP70 detection and measurement of ZAP70 mRNA expression is complicated by the need for T cell depletion. Having identified a correlation between DNA methylation of a 5' regulatory region of ZAP70 and expression of the gene, we and others have proposed the measurement of ZAP70 methylation as an alternative biomarker. Of note, recent work by Claus et al showed ZAP70 methylation to be prognostic for progression free survival (PFS) after therapy in two small, phase II, trial cohorts, treated with regimes incorporating Rituximab. However, as no comprehensive assessment of the prognostic value of ZAP70 methylation has been reported, we analysed ZAP70 methylation in 416 samples from patients entered into the phase III UK LRF CLL4 trial (comparing Chlorambucil(C) with Fludarabine alone (F) or in combination with Cyclophosphamide(FC)). The methylation of a single CpG site 334bp downstream of the transcription start site (C334) was interrogated in peripheral blood mononuclear cell DNA samples, using a quantitative bisulfite pyrosequencing assay. All samples were bisulfite converted, amplified and pyrosequenced in triplicate with a 6 point standard curve in each run. Standard curves for each of the 20 runs were linear (r2 > 0.98) and sensitivity of pyrosequencing was ±5%. C334 methylation ranged from 0-100% and showed a significant inverse correlation with percentage of ZAP70 positive cells, IGHV identity to germline and percentage of CD38 positive cells (all p≤0.001). Methylation levels showed a trimodal, W-shaped, distribution with major modes at 5% and 95% and a minor mode at 50%. Based on this distribution, samples were classified as having Low (C334L 66.6%) methylation ([Figure 1][1]). ![Figure 1][2] Figure 1 Distribution of methylation levels In univariate Kaplan Meier (KM) analysis, no significant difference was seen between C334L and C334Int groups in terms of PFS or OS (median PFS 1.68 versus 1.76 years, p=0.36 and median OS 4.71 years versus 5.41 years, p=0.18). However, C334H cases had a significantly longer PFS and OS than either group (median PFS=2.86 years, median OS=8.60 years, all p
-
ZAP70 methylation is an independent prognostic biomarker for front line therapy of chronic lymphocytic leukemia results from the uk lrf cll4 trial
Blood, 2013Co-Authors: Ben Gregory, Zadie Davis, Monica Else, Jonathan C. Strefford, Daniel Catovsky, Matthew Jj Rosezerilli, David OscierAbstract:In CLL, over-expression of ZAP70 is well established as a prognostic biomarker for unfavourable disease course. However, technical challenges limit the application of flow cytometry for ZAP70 detection and measurement of ZAP70 mRNA expression is complicated by the need for T cell depletion. Having identified a correlation between DNA methylation of a 5' regulatory region of ZAP70 and expression of the gene, we and others have proposed the measurement of ZAP70 methylation as an alternative biomarker. Of note, recent work by Claus et al showed ZAP70 methylation to be prognostic for progression free survival (PFS) after therapy in two small, phase II, trial cohorts, treated with regimes incorporating Rituximab. However, as no comprehensive assessment of the prognostic value of ZAP70 methylation has been reported, we analysed ZAP70 methylation in 416 samples from patients entered into the phase III UK LRF CLL4 trial (comparing Chlorambucil(C) with Fludarabine alone (F) or in combination with Cyclophosphamide(FC)). The methylation of a single CpG site 334bp downstream of the transcription start site (C334) was interrogated in peripheral blood mononuclear cell DNA samples, using a quantitative bisulfite pyrosequencing assay. All samples were bisulfite converted, amplified and pyrosequenced in triplicate with a 6 point standard curve in each run. Standard curves for each of the 20 runs were linear (r2 > 0.98) and sensitivity of pyrosequencing was ±5%. C334 methylation ranged from 0-100% and showed a significant inverse correlation with percentage of ZAP70 positive cells, IGHV identity to germline and percentage of CD38 positive cells (all p≤0.001). Methylation levels showed a trimodal, W-shaped, distribution with major modes at 5% and 95% and a minor mode at 50%. Based on this distribution, samples were classified as having Low (C334L 66.6%) methylation ([Figure 1][1]). ![Figure 1][2] Figure 1 Distribution of methylation levels In univariate Kaplan Meier (KM) analysis, no significant difference was seen between C334L and C334Int groups in terms of PFS or OS (median PFS 1.68 versus 1.76 years, p=0.36 and median OS 4.71 years versus 5.41 years, p=0.18). However, C334H cases had a significantly longer PFS and OS than either group (median PFS=2.86 years, median OS=8.60 years, all p<0.001). C334H cases were significantly enriched with good risk features such as the presence of a 13q deletion, absence of 11q deletion, absence of NOTCH1 mutation, ZAP70 negativity, CD38 negativity and IGHV mutated status (all p<0.02). However, C334H status retained significant prognostic value for PFS and OS in KM analysis when cases were stratified by each of these good risk features. Furthermore, in multivariate Cox regression analysis, C334H cases showed a significantly reduced risk of progression compared to all other cases (HR=0.67 (95%CI 0.48-0.95), p=0.023), independent of treatment received, IGHV status and 11q and 17p deletion status ([Table 1][3]). View this table: Table 1 Multivariate Analysis of Progression Free Survival By identifying a group of patients with hypermethylation of ZAP70 , who display a superior outcome with first line chemotherapy, our results confirm the prognostic value of ZAP70 methylation and show it to be superior to ZAP70 expression as measured by flow cytometry. The independent prognostic value of this, compared with established biomarkers, together with the ease and low cost of the assay, warrant its validation in other large trial cohorts. Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: pending:yes [3]: #T1
-
cross talk between dna methylation and active histone modifications regulates aberrant expression of ZAP70 in cll
Journal of Cellular and Molecular Medicine, 2012Co-Authors: Shilu Amin, David Oscier, Anton Parker, Meagan Walsh, Caroline L Wilson, Elaine Willmore, Derek A Mann, Jelena MannAbstract:Zeta-associated protein of 70 kD (ZAP70) is a recognized adverse prognostic marker in chronic lymphocytic leukaemia (CLL) associated with enhanced B-cell receptor signalling, significantly more aggressive disease course and poor overall survival. Zeta-associated protein of 70 kD is ordinarily expressed in T cells where it has a crucial role in T-cell receptor signalling, whereas its aberrant expression in CLL leads to enhanced B-cell receptor signalling and significantly more aggressive disease course. Although much is known about the activation of ZAP70 following engagement of T-cell receptor, there are little data on the regulation of ZAP70 gene expression in normal T cells or CLL. To understand the molecular events underpinning epigenetic regulation of ZAP70 gene in CLL, we have defined ZAP70 promoter region and outlined the regions crucial in regulating the gene activity. Following a direct comparison of ZAP70+ and ZAP70− primary CLLs, we show ZAP70 promoter in expressing CLLs to be associated with a spectrum of active histone modifications, some of which are tightly linked to aberrant DNA methylation in CLL. Cross-talk between histone modifications and reduced DNA methylation culminates in transcriptional de-repression of ZAP70. Moreover, treatment with histone deacetylase (HDAC) and DNA methylation inhibitors results in recovery of ZAP70 expression, which provides a possible explanation for the failure of HDAC inhibitors in CLL treatment and might serve as a cautionary warning for their future use in treatment of this leukaemia.
-
Cross‐talk between DNA methylation and active histone modifications regulates aberrant expression of ZAP70 in CLL
Journal of Cellular and Molecular Medicine, 2012Co-Authors: Shilu Amin, David Oscier, Anton Parker, Meagan Walsh, Caroline L Wilson, Elaine Willmore, Derek A Mann, Jelena MannAbstract:Zeta-associated protein of 70 kD (ZAP70) is a recognized adverse prognostic marker in chronic lymphocytic leukaemia (CLL) associated with enhanced B-cell receptor signalling, significantly more aggressive disease course and poor overall survival. Zeta-associated protein of 70 kD is ordinarily expressed in T cells where it has a crucial role in T-cell receptor signalling, whereas its aberrant expression in CLL leads to enhanced B-cell receptor signalling and significantly more aggressive disease course. Although much is known about the activation of ZAP70 following engagement of T-cell receptor, there are little data on the regulation of ZAP70 gene expression in normal T cells or CLL. To understand the molecular events underpinning epigenetic regulation of ZAP70 gene in CLL, we have defined ZAP70 promoter region and outlined the regions crucial in regulating the gene activity. Following a direct comparison of ZAP70+ and ZAP70− primary CLLs, we show ZAP70 promoter in expressing CLLs to be associated with a spectrum of active histone modifications, some of which are tightly linked to aberrant DNA methylation in CLL. Cross-talk between histone modifications and reduced DNA methylation culminates in transcriptional de-repression of ZAP70. Moreover, treatment with histone deacetylase (HDAC) and DNA methylation inhibitors results in recovery of ZAP70 expression, which provides a possible explanation for the failure of HDAC inhibitors in CLL treatment and might serve as a cautionary warning for their future use in treatment of this leukaemia.
-
epigenetic regulation of ZAP70 in chronic lymphocytic leukemia
Blood, 2008Co-Authors: Anton Parker, Shilu Amin, Jelena Mann, Ben Gregory, Tricia Zwiefelhofer, Helen White, Oliver Giles Best, Nick Cross, Mathias Ehrich, David OscierAbstract:ZAP70 expression has been shown to be involved in enhanced signalling and more aggressive disease in a subset of CLL. Mechanisms regulating ZAP70 expression are unknown. We have shown previously that despite the absence of a 5’ CpG island, the methylation status of a small region of CpG dinucleotides (CpGs) correlates with the transcriptional state of the gene in both normal lymphocytes and B cell leukemias. Quantitative methylation analysis of 605 CpGs across the 28kb genomic region spanning ZAP70 was performed by MassARRAY on a panel of 17 CLL tumor cell samples, 4 lymphoid cell lines and B cell, T cell and myeloid cell samples pooled from 3 normal individuals. All samples showed hypermethylation of the gene body and of the gene’s two 3’ CpG islands. However, there was variability between samples in the methylation of 12 consecutive CpGs within a 1kb predicted promoter region (PPR), spanning the transcription start site (TSS) and in the methylation of 24 consecutive CpGs in an adjacent 1kb differentially methylated region (DMR), downstream of the TSS. The methylation of the PPR and DMR, together with the expression status of the samples, suggested four different states for the gene (Table 1). Table 1 - ZAP70 gene states defined by ZAP70 expression status and methylation of the PPR and DMR. Bisulphite cloning and sequencing of a PCR amplicon spanning an exon1 C/A SNP (rs2276645) and the PPR/DMR junction was performed together with cDNA pyrosequencing of rs2276645 on the five CLL tumor samples identified with gene state III. All samples showed allele specific methylation (ASM) of the A allele within the DMR and almost complete restriction of ZAP70 expression to the hypomethylated C allele. Bisulphite pyrosequencing of two DMR CpGs in purified leukocyte populations from these cases showed that ASM appears restricted to CLL cells, with hypermethylation and hypomethylation of the myeloid and T cells respectively (Table2). This suggests that while methylation of the DMR is sufficient for allele restriction, ASM does not result from imprinting and may be restricted to CLL tumor cells. Table 2 – Mean methylation of 2 DMR CpGs in leukocyte populations from CLL patients with known ASM of the DMR in their tumor cells. Native chromatin immunoprecipitation (N-ChIP) using anti-AcH3, H3K14Ac and H3K14Me2 antibodies was performed on the 4 cell lines and tumor cells from CLLs 1, 2, 6, 7, 13 and 14 from the MassARRAY series. PCR for amplicons across the PPR and DMR showed the presence of all 3 histone modifications in ZAP70 expressing JURKAT and NALM6 cells but these modifications were absent in the ZAP70 negative NAMALWA and HBL2 cells. In contrast, all 6 CLL samples showed enrichment for all 3 modifications, regardless of gene state, suggesting an open, active/permissive chromatin structure, despite clear differences in methylation of the DMR. Further bisulphite pyrosequencing and N-ChIP of NAMALWA and HBL2 cells cultured for 6 days in the presence of 0.5μM Decitibine showed concomitant DMR demethyaltion, increased AcH3 within the DMR and up regulation of ZAP70 expression, all of which were reversed when the drug stimulus was removed. Taken together this data suggests that ZAP70 is regulated by epigenetic mechanisms, with the methylation status of a small DMR playing a key role, sufficient to differentiate the transcriptional activity of two alleles within a single cell. It is apparent that the gene is primed for expression in all CLL cells and that methylation of the DMR is part of the key switching process between active transcription and silencing. The differences in DMR methylation between an individual’s expressing T cells and CLL cells, suggests that differences may exist in the mechanism of regulation between T and B cells and raises the possibility that such differences could be exploited as targets for therapy.
Arthur Weiss - One of the best experts on this subject based on the ideXlab platform.
-
lck promotes ZAP70 dependent lat phosphorylation by bridging ZAP70 to lat
Nature Immunology, 2018Co-Authors: Wanlin Lo, Neel H Shah, Nagib Ahsan, Veronika Horkova, Ondrej Stepanek, Arthur R Salomon, John Kuriyan, Arthur WeissAbstract:T cell–antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and ZAP70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of ZAP70. Lck binds and stabilizes phosho-ZAP70 by using its SH2 domain, and ZAP70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-ZAP70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of ZAP70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling. TCR signaling initiates a signaling cascade involving the kinases Lck and ZAP70 and the adaptor LAT. Weiss and colleagues discover a proline-rich motif in LAT, which facilitates interactions among Lck, LAT and ZAP70 for efficient TCR signaling.
-
destabilizing the autoinhibitory conformation of ZAP70 induces up regulation of inhibitory receptors and t cell unresponsiveness
Journal of Experimental Medicine, 2017Co-Authors: Debra A Cheng, Yiling Chen, Hongerh Liang, Arthur WeissAbstract:ZAP70 plays a critical role in normal T cell development and T cell function. However, little is known about how perturbation of allosteric autoinhibitory mechanisms in ZAP70 impacts T cell biology. Here, we analyze mice with a hypermorphic ZAP70 mutation, W131A, which destabilizes the autoinhibitory conformation of ZAP70, rendering the kinase in a semiactive state. W131A mutant mice with wild-type T cell receptor (TCR) repertoires exhibited relatively normal T cell development. However, crossing the W131A mutant mice to OTII TCR transgenic mice resulted in increased negative selection of OTII+ thymocytes and in increased thymic and peripheral T regulatory cells. Strikingly, increased basal TCR signaling was associated with a marked increase in inhibitory receptor expression and with T cells that were relatively refractory to TCR stimulation. PD-1 inhibitory receptor blockade partially reversed T cell unresponsiveness. Collectively, disruption of normal ZAP70 autoinhibition engaged negative feedback mechanisms by which negative selection and inhibitory receptors restrain TCR signaling to enforce both central and peripheral tolerance.
-
phosphorylation of a tyrosine residue on ZAP70 by lck and its subsequent binding via an sh2 domain may be a key gatekeeper of t cell receptor signaling in vivo
Molecular and Cellular Biology, 2016Co-Authors: Peter A Thill, Arthur Weiss, Arup K ChakrabortyAbstract:The initiation of signaling in T lymphocytes in response to the binding of the T cell receptor (TCR) to cognate ligands is a key step in the emergence of adaptive immune responses. Conventional models posit that TCR signaling is initiated by the phosphorylation of receptor-associated immune receptor activation motifs (ITAMs). The cytoplasmic tyrosine kinase ZAP70 binds to phosphorylated ITAMs, is subsequently activated, and then propagates downstream signaling. While evidence for such models is provided by experiments with cell lines, in vivo, ZAP70 is bound to phosphorylated ITAMs in resting T cells. However, ZAP70 is activated only upon TCR binding to cognate ligand. We report the results of computational studies of a new model for the initiation of TCR signaling that incorporates these in vivo observations. Importantly, the new model is shown to allow better and faster TCR discrimination between self-ligands and foreign ligands. The new model is consistent with many past experimental observations, and experiments that could further test the model are proposed.
-
ZAP70 is essential for long term survival of naive cd8 t cells
Journal of Immunology, 2014Co-Authors: Ina Schim Van Der Loeff, Manoj Saini, Arthur Weiss, Benedict SeddonAbstract:Survival of naive T cells requires engagement of TCR with self-peptide major histocompatibility Ags. The signaling pathways required to transmit this survival signal are poorly understood. In this study, we asked whether the tyrosine kinase ZAP70 is required to transmit survival signals in naive CD8 T cells. In the absence of ZAP70 expression, thymic development is completely blocked. Using a tetracycline-inducible ZAP70 transgene (TetZAP70), thymic development of ZAP70-deficient TCR transgenic F5 mice was restored. Feeding mice doxycycline to induce ZAP70 expression resulted in repopulation of the peripheral naive compartment. ZAP70 transgene expression was then ablated by withdrawal of doxycycline. Survival of ZAP70-deficient naive CD8 T cells depended on host environment. In hosts with a replete T cell compartment, naive T cells died rapidly in the absence of ZAP70 expression. In lymphopenic hosts, ZAP70-deficient T cells survived far longer, in an IL-7–dependent manner, but failed to undergo lymphopenia-induced proliferation. Analyzing mixed bone marrow chimeras revealed that intact ZAP70-dependent signaling was important for integration of recent thymic emigrants into the mature naive compartment. Finally, we asked whether adaptor function conferred by ZAP70 tyrosines 315 and 319 was necessary for transmission of homeostatic TCR signals. This was done by analyzing F5 mice expressing mutant ZAP70 in which these residues had been mutated to alanines (ZAP70 YYAA ). Inducible ZAP70 expression rescued thymic development in F5 TetZAP70 ZAP70 YYAA mice. However, in the absence of wild-type ZAP70 expression, the ZAP70 YYAA mutant failed to transmit either survival or proliferative homeostatic signals.
-
quantitative and temporal requirements revealed for ZAP70 catalytic activity during t cell development
Nature Immunology, 2014Co-Authors: Byron B Auyeung, Heather J Melichar, Jenny O Ross, Debra A Cheng, Julie Zikherman, Kevan M Shokat, Ellen A Robey, Arthur WeissAbstract:TCR ligation activates the kinase ZAP70. Weiss and colleagues, utilizing 'analog-sensitive' ZAP70 cells, show that thymocyte positive and negative selection exhibit differential temporal requirements for ZAP70 signaling.
Laurence Lagneaux - One of the best experts on this subject based on the ideXlab platform.
-
gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mrna
Haematologica, 2009Co-Authors: Basile Stamatopoulos, Benjamin Haibekains, Carole Equeter, Nathalie Meuleman, Anne Soree, Cecile De Bruyn, Delphine Hanosset, Dominique Bron, Philippe Martiat, Laurence LagneauxAbstract:Background Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. Design and Methods In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. Results Twenty-seven genes were differentially expressed with an FDR<10%, and several genes were significant predictors of treatment-free survival (TFS) and/or overall survival; PDE8A and FCRL family genes (down-regulated in ZAP70+ patients) could predict TFS and overall survival; ITGA4 mRNA (up-regulated in ZAP70+ patients) could significantly predict overall survival. Importantly, gene set enrichment analysis revealed overrepresentation of adhesion/migration genes. We therefore investigated in vitro adhesion/migration capacity of chronic lymphocytic leukemia cells into a stromal microenvironment or in response to conditioned medium. We showed that ZAP70+ cells had better adhesion/migration capacities and only ZAP70+ patient cells responded to microenvironment contact by CXCR4 downregulation. Conclusions We concluded that several prognostic factors are the reflection of microenvironment interactions and that the increased adhesion/migratory capacity of ZAP70+ cells in their microenvironment can explain their better survival and thus the aggressiveness of the disease.
-
Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA.
Haematologica, 2009Co-Authors: Basile Stamatopoulos, Carole Equeter, Nathalie Meuleman, Anne Soree, Cecile De Bruyn, Delphine Hanosset, Dominique Bron, Philippe Martiat, Benjamin Haibe-kains, Laurence LagneauxAbstract:Background Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. Design and Methods In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. Results Twenty-seven genes were differentially expressed with an FDR
-
quantification of ZAP70 mrna in b cells by real time pcr is a powerful prognostic factor in chronic lymphocytic leukemia
Clinical Chemistry, 2007Co-Authors: Basile Stamatopoulos, Benjamin Haibekains, Nathalie Meuleman, Dominique Bron, Philippe Martiat, Hughes Duvillier, Martine Massy, Laurence LagneauxAbstract:Background: Chronic lymphocytic leukemia (CLL) is heterogeneous with respect to prognosis and clinical outcome. The mutational status of the immunoglobulin variable heavy chain region (IGHV) has been used to classify patients into 2 groups in terms of overall survival (OS) and clinical characteristics, but the labor-intensive nature and the cost of this time-consuming analysis has prompted investigations of surrogate markers. Methods: We developed a standardized quantitative real-time reverse transcription-PCR (qPCR) method to measure zeta-chain (TCR)-associated protein kinase (ZAP70) mRNA in purified CD19+ cells. We evaluated this and other methods (flow cytometry analyses of ZAP70 and CD38 proteins and qPCR analysis of lipoprotein lipase mRNA) in a cohort of 108 patients (median follow-up, 82 months) to evaluate any associations with IGHV mutational status, OS, and treatment-free survival (TFS). Results: The association between qPCR-measured ZAP70 and IGHV mutational status was statistically significant [χ2 (1) = 50.95; P <0.0001], and the value of Cramer’s V statistic (0.72) indicated a very strong relation. This method also demonstrated sensitivity, specificity, and positive and negative predictive values of 87.8%, 85.7%, 87.5%, and 86%, respectively. ZAP70 expression was significantly associated with OS ( P = 0.0021) and TFS ( P <0.0001). ZAP70+ patients had significantly shorter median TFS (24 months) than ZAP70− patients (157 months) ( P <0.0001). Moreover, qPCR-measured ZAP70 expression has greater prognostic power than IGHV mutational status and the other prognostic markers tested. Conclusions: ZAP70 mRNA quantification via qPCR is a strong surrogate marker of IGHV mutational status and a powerful prognostic factor.
-
Quantification of ZAP70 mRNA in B cells by real-time PCR is a powerful prognostic factor in chronic lymphocytic leukemia.
Clinical Chemistry, 2007Co-Authors: Basile Stamatopoulos, Nathalie Meuleman, Dominique Bron, Philippe Martiat, Hughes Duvillier, Martine Massy, Benjamin Haibe-kains, Laurence LagneauxAbstract:Background: Chronic lymphocytic leukemia (CLL) is heterogeneous with respect to prognosis and clinical outcome. The mutational status of the immunoglobulin variable heavy chain region (IGHV) has been used to classify patients into 2 groups in terms of overall survival (OS) and clinical characteristics, but the labor-intensive nature and the cost of this time-consuming analysis has prompted investigations of surrogate markers. Methods: We developed a standardized quantitative real-time reverse transcription-PCR (qPCR) method to measure zeta-chain (TCR)-associated protein kinase (ZAP70) mRNA in purified CD19+ cells. We evaluated this and other methods (flow cytometry analyses of ZAP70 and CD38 proteins and qPCR analysis of lipoprotein lipase mRNA) in a cohort of 108 patients (median follow-up, 82 months) to evaluate any associations with IGHV mutational status, OS, and treatment-free survival (TFS). Results: The association between qPCR-measured ZAP70 and IGHV mutational status was statistically significant [χ2 (1) = 50.95; P
Basile Stamatopoulos - One of the best experts on this subject based on the ideXlab platform.
-
gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mrna
Haematologica, 2009Co-Authors: Basile Stamatopoulos, Benjamin Haibekains, Carole Equeter, Nathalie Meuleman, Anne Soree, Cecile De Bruyn, Delphine Hanosset, Dominique Bron, Philippe Martiat, Laurence LagneauxAbstract:Background Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. Design and Methods In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. Results Twenty-seven genes were differentially expressed with an FDR<10%, and several genes were significant predictors of treatment-free survival (TFS) and/or overall survival; PDE8A and FCRL family genes (down-regulated in ZAP70+ patients) could predict TFS and overall survival; ITGA4 mRNA (up-regulated in ZAP70+ patients) could significantly predict overall survival. Importantly, gene set enrichment analysis revealed overrepresentation of adhesion/migration genes. We therefore investigated in vitro adhesion/migration capacity of chronic lymphocytic leukemia cells into a stromal microenvironment or in response to conditioned medium. We showed that ZAP70+ cells had better adhesion/migration capacities and only ZAP70+ patient cells responded to microenvironment contact by CXCR4 downregulation. Conclusions We concluded that several prognostic factors are the reflection of microenvironment interactions and that the increased adhesion/migratory capacity of ZAP70+ cells in their microenvironment can explain their better survival and thus the aggressiveness of the disease.
-
Gene expression profiling reveals differences in microenvironment interaction between patients with chronic lymphocytic leukemia expressing high versus low ZAP70 mRNA.
Haematologica, 2009Co-Authors: Basile Stamatopoulos, Carole Equeter, Nathalie Meuleman, Anne Soree, Cecile De Bruyn, Delphine Hanosset, Dominique Bron, Philippe Martiat, Benjamin Haibe-kains, Laurence LagneauxAbstract:Background Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia, but mechanisms by which its higher expression leads to a poor outcome must still be fully explained. Design and Methods In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B cells from chronic lymphocytic leukemia patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Two groups of 7 patients were compared, selected on the basis of either high or low ZAP70 mRNA expression. Results Twenty-seven genes were differentially expressed with an FDR
-
quantification of ZAP70 mrna in b cells by real time pcr is a powerful prognostic factor in chronic lymphocytic leukemia
Clinical Chemistry, 2007Co-Authors: Basile Stamatopoulos, Benjamin Haibekains, Nathalie Meuleman, Dominique Bron, Philippe Martiat, Hughes Duvillier, Martine Massy, Laurence LagneauxAbstract:Background: Chronic lymphocytic leukemia (CLL) is heterogeneous with respect to prognosis and clinical outcome. The mutational status of the immunoglobulin variable heavy chain region (IGHV) has been used to classify patients into 2 groups in terms of overall survival (OS) and clinical characteristics, but the labor-intensive nature and the cost of this time-consuming analysis has prompted investigations of surrogate markers. Methods: We developed a standardized quantitative real-time reverse transcription-PCR (qPCR) method to measure zeta-chain (TCR)-associated protein kinase (ZAP70) mRNA in purified CD19+ cells. We evaluated this and other methods (flow cytometry analyses of ZAP70 and CD38 proteins and qPCR analysis of lipoprotein lipase mRNA) in a cohort of 108 patients (median follow-up, 82 months) to evaluate any associations with IGHV mutational status, OS, and treatment-free survival (TFS). Results: The association between qPCR-measured ZAP70 and IGHV mutational status was statistically significant [χ2 (1) = 50.95; P <0.0001], and the value of Cramer’s V statistic (0.72) indicated a very strong relation. This method also demonstrated sensitivity, specificity, and positive and negative predictive values of 87.8%, 85.7%, 87.5%, and 86%, respectively. ZAP70 expression was significantly associated with OS ( P = 0.0021) and TFS ( P <0.0001). ZAP70+ patients had significantly shorter median TFS (24 months) than ZAP70− patients (157 months) ( P <0.0001). Moreover, qPCR-measured ZAP70 expression has greater prognostic power than IGHV mutational status and the other prognostic markers tested. Conclusions: ZAP70 mRNA quantification via qPCR is a strong surrogate marker of IGHV mutational status and a powerful prognostic factor.
-
Quantification of ZAP70 mRNA in B cells by real-time PCR is a powerful prognostic factor in chronic lymphocytic leukemia.
Clinical Chemistry, 2007Co-Authors: Basile Stamatopoulos, Nathalie Meuleman, Dominique Bron, Philippe Martiat, Hughes Duvillier, Martine Massy, Benjamin Haibe-kains, Laurence LagneauxAbstract:Background: Chronic lymphocytic leukemia (CLL) is heterogeneous with respect to prognosis and clinical outcome. The mutational status of the immunoglobulin variable heavy chain region (IGHV) has been used to classify patients into 2 groups in terms of overall survival (OS) and clinical characteristics, but the labor-intensive nature and the cost of this time-consuming analysis has prompted investigations of surrogate markers. Methods: We developed a standardized quantitative real-time reverse transcription-PCR (qPCR) method to measure zeta-chain (TCR)-associated protein kinase (ZAP70) mRNA in purified CD19+ cells. We evaluated this and other methods (flow cytometry analyses of ZAP70 and CD38 proteins and qPCR analysis of lipoprotein lipase mRNA) in a cohort of 108 patients (median follow-up, 82 months) to evaluate any associations with IGHV mutational status, OS, and treatment-free survival (TFS). Results: The association between qPCR-measured ZAP70 and IGHV mutational status was statistically significant [χ2 (1) = 50.95; P
Benedict Seddon - One of the best experts on this subject based on the ideXlab platform.
-
a ZAP70 dependent feedback circuit is essential for efficient selection of cd4 lineage thymocytes
Immunology and Cell Biology, 2015Co-Authors: Charles Sinclair, Benedict SeddonAbstract:A ZAP70-dependent feedback circuit is essential for efficient selection of CD4 lineage thymocytes
-
ZAP70 is essential for long term survival of naive cd8 t cells
Journal of Immunology, 2014Co-Authors: Ina Schim Van Der Loeff, Manoj Saini, Arthur Weiss, Benedict SeddonAbstract:Survival of naive T cells requires engagement of TCR with self-peptide major histocompatibility Ags. The signaling pathways required to transmit this survival signal are poorly understood. In this study, we asked whether the tyrosine kinase ZAP70 is required to transmit survival signals in naive CD8 T cells. In the absence of ZAP70 expression, thymic development is completely blocked. Using a tetracycline-inducible ZAP70 transgene (TetZAP70), thymic development of ZAP70-deficient TCR transgenic F5 mice was restored. Feeding mice doxycycline to induce ZAP70 expression resulted in repopulation of the peripheral naive compartment. ZAP70 transgene expression was then ablated by withdrawal of doxycycline. Survival of ZAP70-deficient naive CD8 T cells depended on host environment. In hosts with a replete T cell compartment, naive T cells died rapidly in the absence of ZAP70 expression. In lymphopenic hosts, ZAP70-deficient T cells survived far longer, in an IL-7–dependent manner, but failed to undergo lymphopenia-induced proliferation. Analyzing mixed bone marrow chimeras revealed that intact ZAP70-dependent signaling was important for integration of recent thymic emigrants into the mature naive compartment. Finally, we asked whether adaptor function conferred by ZAP70 tyrosines 315 and 319 was necessary for transmission of homeostatic TCR signals. This was done by analyzing F5 mice expressing mutant ZAP70 in which these residues had been mutated to alanines (ZAP70 YYAA ). Inducible ZAP70 expression rescued thymic development in F5 TetZAP70 ZAP70 YYAA mice. However, in the absence of wild-type ZAP70 expression, the ZAP70 YYAA mutant failed to transmit either survival or proliferative homeostatic signals.
-
regulation of ZAP70 expression during thymocyte development enables temporal separation of cd4 and cd8 repertoire selection at different signaling thresholds
Science Signaling, 2010Co-Authors: Manoj Saini, Charles Sinclair, Daniel Marshall, Mauro Tolaini, Shimon Sakaguchi, Benedict SeddonAbstract:To investigate the temporal regulation of the commitment of immature thymocytes to either the CD4 + or the CD8 + lineage in the thymus, we developed a transgenic mouse that expressed a tetracycline-inducible gene encoding the tyrosine kinase ζ chain–associated protein kinase of 70 kD (ZAP70), which restored development in ZAP70 −/− thymocytes arrested at the preselection, CD4 + CD8 + double-positive (DP) stage. After induction of the expression of ZAP70 and the production of ZAP70 protein, CD4 + single-positive (SP) cells that expressed Zbtb7b (which encodes the CD4 + T cell–associated transcription factor ThPOK) became abundant within 30 hours, whereas CD8 + SP cells were not detectable until day 4. We found that mature CD4 + and CD8 + cells arose from phenotypically distinct subsets of DP thymocytes that developed with different kinetics and contrasting sensitivities to stimulation of the T cell antigen receptor (TCR). In wild-type mice, expression of endogenous ZAP70 progressively increased during maturation of the DP subsets, and the abundance of ZAP70 protein determined the sensitivity of the cells to stimulation of the TCR. This temporal gradient in the amount of ZAP70 protein enabled the selection of CD4 + and CD8 + repertoires in separate temporal windows and at different TCR signaling thresholds, thereby facilitating discrimination of distinct positive selection signals in these lineages.
