Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ZAP”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed2004

ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia.

BACKGROUND: The course of chronic lymphocytic leukemia (CLL) is variable. In aggressive disease, the CLL cells usually express an unmutated immunoglobulin heavy-chain variable-region gene (IgV(H)) and the 70-kD zeta-associated protein (ZAP-70), whereas in indolent disease, the CLL cells usually express mutated IgV(H) but lack expression of ZAP-70. METHODS: We evaluated the CLL B cells from 307 patients with CLL for ZAP-70 and mutations in the rearranged IgV(H) gene. We then investigated the association between the results and the time from diagnosis to initial therapy. RESULTS: We found that ZAP-70 was expressed above a defined threshold level in 117 of the 164 patients with an unmutated IgV(H) gene (71 percent), but in only 24 of the 143 patients with a mutated IgV(H) gene (17 percent, P<0.001). Among the patients with ZAP-70-positive CLL cells, the median time from diagnosis to initial therapy in those who had an unmutated IgV(H) gene (2.8 years) was not significantly different from the median time in those who had a mutated IgV(H) gene (4.2 years, P=0.07). However, the median time from diagnosis to initial treatment in each of these groups was significantly shorter than the time in patients with ZAP-70-negative CLL cells who had either mutated or unmutated IgV(H) genes (P<0.001). The median time from diagnosis to initial therapy among patients who did not have ZAP-70 was 11.0 years in those with a mutated IgV(H) gene and 7.1 years in those with an unmutated IgV(H) gene (P<0.001). CONCLUSIONS: Although the presence of an unmutated IgV(H) gene is strongly associated with the expression of ZAP-70, ZAP-70 is a stronger predictor of the need for treatment in B-cell CLL.

B-Lymphocytes↗
PubMed1996

Differential intrinsic enzymatic activity of Syk and Zap-70 protein-tyrosine kinases.

Syk and Zap-70 are related protein-tyrosine kinases implicated in antigen and Fc receptor signaling. While Zap-70 is restricted to T-cells and natural killer cells, Syk accumulates in B-cells, mast cells, platelets, and immature T-cells. In addition, we found that an isoform of Syk (SykB), which carries a 23-amino acid deletion in the "linker" region, is prominently expressed in bone marrow. To better understand the relative impact of Syk, SykB, and Zap-70 on signal transduction, we compared their intrinsic enzymatic properties in transiently transfected COS-1 cells and in hemopoietic cells. Using modified versions of these enzymes bearing a common Myc epitope at the amino terminus, we determined that the ability of Syk and SykB to undergo autophosphorylation and to phosphorylate erythrocyte band 3 in immune complex kinase reactions was at least 100-fold greater than that of Zap-70. Similarly, Syk and SykB, but not Zap-70, caused prominent tyrosine phosphorylation of p120(c-)cbl in COS-1 cells. A similar pattern of activity was also noted for endogenous Syk and Zap-70 from hemopoietic cells. To understand the structural basis for these characteristics, we also created and analyzed a series of chimeras between Syk and Zap-70. These studies indicated that the catalytic domain of Syk and Zap-70, but not their SH2 domains, linker region or carboxyl-terminal tail, was responsible for their respective activity. Taken together, these data demonstrated that the intrinsic enzymatic activity of Syk and SykB is superior to that of Zap-70 and that such a distinction relates to structural variations in the catalytic domain.

Amino Acid Sequence↗
PubMed1996

Mutation of tyrosines 492/493 in the kinase domain of ZAP-70 affects multiple T-cell receptor signaling pathways.

The protein-tyrosine kinase ZAP-70 is implicated, together with the Src kinase p56(lck), in controlling the early steps of the T-cell antigen receptor (TCR) signaling cascade. To help elucidate further the mechanism by which ZAP-70 regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited TCR-induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp36-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wild-type ZAP-70 induced constitutive activation of nuclear factor of activated T cells. The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR zeta chain and was able to bind p56(lck). This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain 2-mediated association of p56(lck) with ZAP-70. Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56(lck), which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling.

Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivo capacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT. Collectively, our data reveal the pivotal role of Tyr319 phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling.

Amino Acid Sequence↗
PubMed1999

Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck.

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.

Amino Acid Sequence↗
PubMed1999

T cell activation stimulates the association of enzymatically active tyrosine-phosphorylated ZAP-70 with the Crk adapter proteins.

Engagement of the T cell antigen receptor initiates signal transduction involving tyrosine phosphorylation of multiple effector molecules and the formation of multimolecular complexes at the receptor site. Adapter proteins that possess SH2 and SH3 protein-protein interaction domains are implicated in the assembly of cell activation-induced signaling complexes. We found that Crk adapter proteins undergo activation-induced interaction with the zeta-chain associated protein (ZAP-70) tyrosine kinase in the human T cell line, Jurkat. Incubation of various glutathione S-transferase fusion proteins with a lysate of activated Jurkat cells resulted in selective association of ZAP-70 with Crk, but not Grb2 or Nck, adapter proteins. In addition, tyrosine-phosphorylated ZAP-70 co-immunoprecipitated with Crk from a lysate of activated Jurkat cells, and ZAP-70 association with GST-Crk was observed in a lysate of activated human peripheral blood T cells. Association between the two molecules was mediated by direct physical interaction and involved the Crk-SH2 domain and phosphotyrosyl-containing sequences on ZAP-70. The association required intact Lck, considered to be an upstream regulator of ZAP-70, because it could not take place in activated JCaM1 cells, which express normal levels of ZAP-70 but are devoid of Lck. Finally, glutathione S-transferase-Crk fusion proteins were found to interact predominantly with membrane-residing tyrosine-phosphorylated ZAP-70 that exhibited autophosphorylation activity as well as phosphorylation of an exogenous substrate, CFB3. These findings suggest that Crk adapter proteins play a role in the early activation events of T lymphocytes, apparently, by direct interaction with, and regulation of, the membrane-residing ZAP-70 protein tyrosine kinase.

Adaptor Proteins, Signal Transducing↗
PubMed1998

ZAP-70 tyrosine kinase is required for LFA-1-dependent T cell migration.

The ZAP-70 tyrosine kinase is essential for T cell activation by the T cell receptor. We show that ZAP-70 is also required for migration of T cells that is dependent on the integrin LFA-1. Invasion of TAM2D2 T cell hybridoma cells into fibroblast monolayers, which is LFA-1-dependent, was blocked by overexpression of dominant-negative ZAP-70 and by piceatannol but not by herbimycin A. The Syk inhibitor piceatannol blocks the Syk homologue ZAP-70, which is expressed by TAM2D2 cells, with the same dose dependence as the inhibition of invasion. Dominant-negative ZAP-70 completely inhibited the extensive metastasis formation of TAM2D2 cells to multiple organs upon i.v. injection into mice. Migration of TAM2D2 cells through filters coated with the LFA-1 ligand ICAM-1, induced by 1 ng/ml of the chemokine SDF-1, was blocked by anti-LFA-1 mAb and also abrogated by dominant-negative ZAP-70 and piceatannol. In contrast, migration induced by 100 ng/ml SDF-1 was independent of both LFA-1 and ZAP-70. LFA-1 cross-linking induced tyrosine phosphorylation, which was blocked by dominant-negative ZAP-70 and piceatannol. We conclude that LFA-1 engagement triggers ZAP-70 activity that is essential for LFA-1-dependent migration.

Animals↗
PubMed2006

ZAP-70 mRNA quantification in B-cell chronic lymphocytic leukaemia.

OBJECTIVE: The mutational status of the immunoglobulin (Ig) V(H) gene in B-cell chronic lymphocytic leukaemia (B-CLL) identifies two subgroups of patients with significantly different outcomes. We investigated the association of ZAP-70 expression with IgVH mutational status in B-CLL by quantifying ZAP-70 mRNA, to evaluate its use as a surrogate marker for mutational status. The aim of this study was to develop a quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR) assay for the detection of ZAP-70 expression in a group of patients whose mutational status and cytogenetics had been determined previously. METHODS: RQ-PCR was used to analyse ZAP-70 expression from 42 B-CLL patients. B cells were purified using CD19 magnetic bead system and total RNA was isolated. RQ-PCR was performed using Taqman PCR. RESULTS: Twenty-five patients (60%) had mutated and 17 (40%) had unmutated IgVH genes; 94% (16/17) of patients with unmutated IgVH gene were ZAP-70 positive as assessed by RQ-PCR and 92% (23/25) of patients with mutated IgVH gene were ZAP-70 negative. In three patients, ZAP-70 expression and IgVH mutational status were discordant. CONCLUSION: This paper describes an RQ-PCR assay for the detection of ZAP-70 expression and confirms that IgV(H) unmutated CLL cells have a high expression of ZAP-70 in comparison with IgVH mutated CLL. This robust method acts as a surrogate marker for IgVH mutational status albeit with <100% concordance. However, it does provide better concordance with mutational status than that reported using flow cytometry.

Adult↗
PubMed2003

ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile.

The presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IgVH) of chronic lymphocytic leukemia (CLL) cells provides prognostic information. Patients whose leukemic cells express unmutated IgVH regions (Ig-unmutated CLL) often have progressive disease, whereas patients whose leukemic cells express mutated IgVH regions (Ig-mutated CLL) more often have an indolent disease. Given the difficulty in performing IgVH sequencing in a routine diagnostic laboratory, this prognostic distinction is currently unavailable to most patients. Pilot gene expression profiling studies in patients with CLL identified genes that were differentially expressed between the Ig-unmutated and Ig-mutated CLL subtypes. Here, we have profiled an expanded cohort of 107 patients and show that ZAP-70 is the gene that best distinguishes the CLL subtypes. Ig-unmutated CLL expressed ZAP-70 5.54-fold more highly than Ig-mutated CLL (P < 10(-21)). ZAP-70 expression correctly predicted IgVH mutation status in 93% of patients. ZAP-70 expression and IgVH mutation status were comparable in their ability to predict time to treatment requirement following diagnosis. In 7 patients, ZAP-70 expression and IgVH mutation status were discordant: 4 Ig-mutated CLLs had high ZAP-70 expression and 3 Ig-unmutated CLLs had low ZAP-70 expression. Among these ZAP-70 "outliers," those with Ig-mutated CLL had clinical features that are uncharacteristic of this CLL subtype: 2 required early treatment and 2 used a mutated VH3-21 gene, an IgVH gene that has been associated with progressive disease. We developed reverse transcriptase-polymerase chain reaction and immunohistochemical assays for ZAP-70 expression that can be applied clinically and would yield important prognostic information for patients with CLL.

ADP-ribosyl Cyclase↗
PubMed2005

ZAP-70 expression is associated with enhanced ability to respond to migratory and survival signals in B-cell chronic lymphocytic leukemia (B-CLL).

Molecular markers like IgV(H) mutational status, chromosomal abnormalities, and CD38 and ZAP-70 expression have prognostic value in B-cell chronic lymphocytic leukemia (B-CLL). These may be pathogenetic because of the coincidental expression of ZAP-70 and increased B-cell receptor (BCR) signaling and the signaling function of CD38 in CLL. This study shows that ZAP-70(+) CLL B cells respond in vitro more readily than ZAP-70(-) CLL and normal B cells to chemokine migratory signals through enhanced surface CCR7 expression (P = .009; P < .001) and increased responsiveness to its ligands CCL19 and CCL21, demonstrated by F-actin polymerization (P < .05) and cellular migration (P < .01). In addition, ZAP-70(+) CLL cells exhibit sustained ERK phosphorylation/activation following stimulation with CXCL12 (SDF1-alpha, a survival factor produced by stromal cells) compared with ZAP-70(-) cells (P = .004). Following coculture with nurse-like cells, the survival of ZAP-70(+) but not ZAP-70(-) CLL cells is significantly enhanced by the addition of CXCL12 (P < .05), an effect that is partially blocked by the MEK inhibitor PD98059. These advantageous migratory and survival responses may promote easier access to and greater proliferation in pseudo-germinal centers and explain in part the more progressive nature of ZAP-70(+) disease.

Case-Control Studies↗
PubMed2005

ZAP-70 expression in acute lymphoblastic leukemia: association with the E2A/PBX1 rearrangement and the pre-B stage of differentiation and prognostic implications.

We evaluated the expression of 2 members of the Syk family, ZAP-70 and Syk, in acute lymphoblastic leukemia (ALL) samples, using data derived from a series of 33 T-ALL and 95 B-lineage adult ALL patients analyzed by oligonucleotide arrays. Of the B-lineage ALL cases, 37 were BCR/ABL+, 10 were ALL1/AF4+, 5 were E2A/PBX1+, and 43 carried no known molecular abnormality. ZAP-70 was highly expressed in T-ALL. A high ZAP-70 expression was also found in a proportion of B-lineage ALL, the highest levels being associated with the E2A/PBX1+ group and the lowest with ALL1/AF4+ cases (P < .001). A higher ZAP-70 expression was also observed in the pre-B group (P < .001). Remarkably, Syk expression was always preserved, suggesting that ZAP-70 expression is not substitutive of Syk. At the protein level, ZAP-70 was evaluated on 39 newly diagnosed ALL patients (25 adults, 14 children) and was detected in 23 cases (59%). ZAP-70 expression was consistently found in Ig mu+ cases. Evaluation of long-term outcome in cases without molecular abnormalities showed that the higher levels of ZAP-70 were coupled to a higher relapse rate. In ALL, ZAP-70 expression is associated with the E2A/PBX1 rearrangement and pre-B stage and may have a prognostic role and be a candidate molecule for targeted therapies.

Adult↗
PubMed2000

Differential requirement of ZAP-70 for CD2-mediated activation pathways of mature human T cells.

This study addresses the role of the tyrosine kinase ZAP-70 in CD2-mediated T cell activation. Patients lacking ZAP-70 have few mature CD8+ T cells and high numbers of CD4+ T cells that are nonfunctional upon TCR triggering. Such a patient with a homozygous deletion in the zap-70 gene that resulted in the complete absence of ZAP-70 protein expression has been identified. Expression of the tyrosine kinases Lck, Fyn, and Syk was normal. The patient's T cells were activated with two different pairs of mitogenic mAbs. CD2-induced phosphorylation of the zeta-chain and influx of Ca2+ was defective in the ZAP-70-deficient T cells, whereas CD2-induced phosphorylation of several other proteins, including Syk, was not affected. CD2-induced proliferation as well as production of TNF-alpha and IFN-gamma was abrogated in ZAP-70-deficient T cells, whereas PMA plus ionomycin induced normal activation of these cells. Together, this study shows that CD2-activation triggers ZAP-70-dependent and -independent pathways. Deletion of ZAP-70 affected CD2- and CD3-mediated proliferation and cytokine production in a similar way, suggesting that one of the different CD2 pathways converges with a CD3 pathway at or upstream of the activation of ZAP-70.

CD2 Antigens↗
PubMed2001

Distinct T cell developmental consequences in humans and mice expressing identical mutations in the DLAARN motif of ZAP-70.

The protein tyrosine kinase, ZAP-70, is pivotally involved in transduction of Ag-binding signals from the TCR required for T cell activation and development. Defects in ZAP-70 result in SCID in humans and mice. We describe an infant with SCID due to a novel ZAP-70 mutation, comparable with that which arose spontaneously in an inbred mouse colony. The patient inherited a homozygous missense mutation within the highly conserved DLAARN motif in the ZAP-70 kinase domain. Although the mutation only modestly affected protein stability, catalytic function was absent. Despite identical changes in the amino acid sequence of ZAP-70, the peripheral T cell phenotypes of our patient and affected mice are distinct. ZAP-70 deficiency in this patient, as in other humans, is characterized by abundant nonfunctional CD4(+) T cells and absent CD8(+) T cells. In contrast, ZAP-70-deficient mice lack both major T cell subsets. Although levels of the ZAP-70-related protein tyrosine kinase, Syk, may be sufficiently increased in human thymocytes to rescue CD4 development, survival of ZAP-70-deficient T cells in the periphery does not appear to be dependent on persistent up-regulation of Syk expression.

Amino Acid Motifs↗
PubMed1999

Degradation of ZAP-70 following antigenic stimulation in human T lymphocytes: role of calpain proteolytic pathway.

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.

Antigen-Presenting Cells↗
PubMed2000

ZAP-70 is required for calcium mobilization but is dispensable for mitogen-activated protein kinase (MAPK) superfamily activation induced via CD2 in human T cells.

Stimulation with specific pairs of anti-CD2 antibodies can induce T cell activation and proliferation. In this study, we investigate the significance of ZAP-70 in CD2 signaling using ZAP-70-deficient T cells derived from a CD8-deficient patient and show that ZAP-70 is necessary for cellular proliferation and cytokine production in T cells stimulated via CD2. Biochemical analyses show that CD2 stimulation induces activation of mitogen-activated protein kinase (MAPK) superfamily in ZAP-70-deficient T cells, indicating that a ZAP-70-independent pathway(s) exists for MAPK superfamily activation via CD2. In contrast, intracellular Ca(2+) mobilization and activation of nuclear factor of activated T cells (NFAT) upon CD2 triggering were impaired in T cells lacking ZAP-70. Furthermore, we found that pharmacological Ca(2+) elevation combined with CD2 stimulation restored NFAT activation and subsequent cytokine production in ZAP-70-deficient T cells. These results indicate that in CD2 signaling, ZAP-70 plays an essential role in Ca(2+) mobilization and NFAT activation.

Animals↗
PubMed2006

ZAP-70 by flow cytometry: a comparison of different antibodies, anticoagulants, and methods of analysis.

BACKGROUND: The clinical course of chronic lymphocytic leukaemia (CLL) is variable. ZAP-70 expression is believed to provide prognostic information. The flow cytometric detection of ZAP-70 is difficult because it is an intracellular antigen with weak expression in CLL. Consensus has not been reached as to the best method for measurement. METHODS: We analyzed 72 CLL patient samples for ZAP-70 expression and IgVH mutational status. Sensitivity and specificity of ZAP-70 expression against IgVH mutational status were assessed for two clones (2F3.2 and 1E7.2) and for four methods of analysis: percentage positivity (PP), comparing test to isotype control, ratio of geometric means of test and isotype control, and percentage and ratiometric methods comparing test and T/NK cell populations. The effects of anticoagulant, collection times, and time to analysis were also evaluated. RESULTS: Sensitivity and specificity were 85 and 88%, respectively, for Upstate PP; 70 and 88% for Caltag PP; 89 and 91% for Upstate ratio; 89 and 88% for Caltag ratio. Intraobserver variability was smaller when ZAP-70 expression was assessed using a ratiometric approach rather than the percentage method. By 48 h, we observed an average decrease of 13% in the Caltag ratio in the heparin preserved samples compared to an increase of 3% in those collected in EDTA. Within the first 24-h period, a greater percent variability was observed in those samples collected into EDTA compared with heparin. CONCLUSION: Our data support a rapid method for ZAP-70 measurement using commercially available fixation/permeabilization reagents, a conjugated antibody, and a ratiometric method of analysis that minimizes subjective interpretation of the results. This is a method of ZAP-70 assessment that could be included in a routine diagnostic CLL panel; however, the choice of anticoagulant and time of analysis after collection are critical factors in accurate assessment of ZAP-70 expression.

Antibodies↗
PubMed2006

Flow cytometry for ZAP-70: New colors for chronic lymphocytic leukemia.

ZAP-70 has become one of the most studied prognostic markers in Chronic Lymphocytic Leukemia (CLL). ZAP-70 is remarkable in many ways: ZAP-70 has been identified as the best discriminating gene between prognostically distinct CLL subtypes using large scale gene expression profiling; ZAP-70 has been shown to enhance signal transduction in CLL B-cells and therefore could contribute to disease progression; and ZAP-70 is one of the rare examples of an intracellular target considered for clinical flow cytometry. This issue attests to the enormous effort and the steady progress made in overcoming technical challenges of testing for ZAP-70 expression and sets the foundation for a successful translation of this important marker into clinical practice. Despite the best effort, one will likely have to accept that not all cases can be clearly assigned to one or the other group, given that ZAP-70 expression between CLL patients falls along a continuum from absent to high. Nevertheless, ZAP-70 expression could become a key parameter to guide patients towards risk adapted treatment strategies in prospective clinical trials.

Biomarkers, Tumor↗
PubMed2006

Variations in the detection of ZAP-70 in chronic lymphocytic leukemia: Comparison with IgV(H) mutation analysis.

Lack of immunoglobulin heavy chain genes (IgV(H)) mutation in patients with chronic lymphocytic leukemia (CLL) is associated with rapid disease progression and shorter survival. The zeta-chain (T-cell receptor) associated protein kinase 70 kDa (ZAP-70) has been reported to be a surrogate marker for IgV(H) mutation status, and its expression in leukemic cells correlates with unmutated IgV(H). However, ZAP-70 detection by flow cytometry varies significantly dependant on the antibodies used, the method of performing the assay, and the condition of the cells in the specimen. The clinical value of ZAP-70 testing when samples are shipped under poorly controlled conditions is not known. Furthermore, testing in a research environment may differ from testing in a routine clinical laboratory. We validated an assay for ZAP-70 by comparing results with clinical outcome and the mutation status of the IgV(H). Using stored samples, we show significant correlation between ZAP-70 expression and clinical outcome as well as IgV(H) mutation at a cut-off point of 15%. While positive samples (>15% positivity) remain positive when kept in the laboratory environment for 48 h after initial testing, results obtained from samples from CLL patients tested after shipping at room temperature for routine testing showed no correlation with IgV(H) mutation status when 15% cut-off was used. In these samples, cut-point of 10% correlated with the IgV(H) mutation (P = 0.0001). This data suggests that although ZAP-70 positivity correlates with IgV(H) mutation status and survival, variations in sample handling and preparation may influence results. We show that IgV(H) mutation results, unlike ZAP-70 remain correlated with CD38 expression and beta-2 microglobulin in shipped samples, and ZAP-70 testing should not be used as the sole criterion for stratifying patients for therapy.

ADP-ribosyl Cyclase 1↗