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PubMed2006

Comparison of bone marrow and peripheral blood ZAP-70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemia.

BACKGROUND: : The mutational status of the immunoglobulin heavy chain variable gene in patients with chronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare better than those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP-70 to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP-70 expression in tumor cells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a few studies have shown a small number of discordant results between ZAP-70 positivity, IgVH mutational status, and clinical outcome. There have been no reported studies comparing bone marrow samples with peripheral blood for ZAP-70 expression in CLL patients. METHODS: : We searched our flow cytometry files from October 2004 through April 2006 and identified CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP-70 positivity as greater than 30% of the CD19(+) B-cells above the isotype control value that coexpress ZAP-70. Statistical analyses were performed using the Fisher exact test and student t-test. RESULTS: : A significantly greater number of bone marrow specimens were positive for ZAP-70 when compared with the number of peripheral blood specimens. Of all the ZAP-70 negative specimens, CLL cells from bone marrow had a greater mean percentage of ZAP-70 positive cells when compared with the CLL cells from peripheral blood. Finally, six patients were identified who were ZAP-70 positive in the bone marrow but ZAP-70 negative in the peripheral blood. CONCLUSIONS: : These results may be due to either an increase in the false positive rate in bone marrow specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct from peripheral tumor cells. As prognosis and treatment decisions may be based on ZAP-70 results from either specimen type, it is prudent to further examine this observation. (c) 2006 International Society for Analytical Cytology.

Adult↗
PubMed2006

Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process.

The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patient's prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold-standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta-associated protein (ZAP-70) expression was associated with unmutated IgVH genes and ZAP-70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP-70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP-70 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti-ZAP-70 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti-ZAP-70 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP-70 levels were expressed as percentage positive (POS) relative to isotype control or normal B-cells or normal T-cells; in addition the levels were reported as a ratio of expression in CLL cells relative to T-cells. The reported level of ZAP-70 expression varied greatly depending on the antibody and the method used to express the results. The CLL/T-cell ZAP-70 expression ratio showed a much lower interlaboratory variation than other reporting strategies and is recommended for multicenter studies. Stabilization results in decreased expression of CD19 making gating more difficult and therefore stabilized samples are not optimal for multicentric analysis of ZAP-70 expression. We assessed the variation of ZAP-70 expression levels in fresh cells according to storage time, which demonstrated that ZAP-70 is labile but sufficiently stable to allow comparison using fresh samples distributed between labs in Europe. These studies have demonstrated progress toward a consensus reporting procedure, and further work is underway to harmonize the preparation and analysis procedures.

Antibodies, Monoclonal↗
PubMed2006

Bone marrow infiltration pattern in B-cell chronic lymphocytic leukemia is related to immunoglobulin heavy-chain variable region mutation status and expression of 70-kd zeta-associated protein (ZAP-70).

B-cell chronic lymphocytic leukemia (B-CLL) consists of at least 2 subtypes with either somatically mutated or unmutated immunoglobulin heavy-chain variable region (IgVH) genes. A prognostic significance of the infiltration pattern in bone marrow trephine biopsy has been described before. The combined pattern analysis and detection of 70-kd zeta-associated protein (ZAP-70) expression in formalin-fixed, paraffin-embedded bone marrow trephines has not been investigated so far. To evaluate the relationship between ZAP-70 expression, mutation status, and the infiltration pattern in B-CLL, we analyzed bone marrow trephine biopsies from B-CLL patients (n = 35). The expression of ZAP-70 was related to the infiltration type: in all samples with diffuse infiltration pattern, the leukemic cells showed ZAP-70 staining, whereas leukemic cells in a nodular infiltration pattern were negative. By contrast, the mixed-pattern type showed a variable ZAP-70 expression. Besides definitely negative or positive ZAP-70 expression, a few samples showed a faint ZAP-70 staining, and the classification into the positive or negative group was difficult. In addition, the infiltration type was related to the mutation status in a subset of samples: mutation of the IgVH gene was restricted to the nondiffuse infiltration pattern and was not found in cases with diffuse infiltration of bone marrow. The expression of ZAP-70, detected by an immunohistochemical assay and also by real-time quantitative reverse transcriptase-polymerase chain reaction assigned 83% of the chronic lymphocytic leukemia cases to the suspected immunoglobulin mutation subtype. In 2 patients with ZAP-70 expression, a mutated IgVH status was found. These cases exhibited a mixed pattern of infiltration. We conclude that the pure nodular type of marrow infiltration in B-CLL is associated with IgH hypermutation and ZAP-70 negativity, whereas the predominantly diffuse type of infiltration reveals unmutated IgH genes with ZAP-70 overexpression. The mixed type of infiltration is displayed by mutated as well as unmutated cases with a varying pattern of ZAP-70 expression.

Adult↗
PubMed2006

Clinical significance of ZAP-70 protein expression in B-cell chronic lymphocytic leukemia.

The clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is variable, and novel biologic parameters need to be added to the clinical staging systems to predict an indolent or aggressive outcome. We investigated the 70-kDa zeta-associated protein (ZAP-70), CD38, soluble CD23 (sCD23), and cytogenetics in 289 patients with B-CLL. Both a shorter progression-free survival (PFS) and overall survival (OS) were observed in ZAP-70(+) (P < .001), in CD38(+) (P < .001) and in sCD23(+) patients (P < .001 and P = .013, respectively). ZAP-70(+)CD38(+) or ZAP-70(+) patients with an unmutated IgV(H) status showed both a shorter PFS (P < .001) and OS (P < .001 and P < .001, respectively) as compared with ZAP-70(-)/CD38(-) or ZAP-70(-) patients with mutated IgV(H) genes. Discordant patients showed an intermediate outcome. Note, ZAP-70(+) patients even if CD38(-) or mutated showed a shorter PFS, whereas ZAP-70(-) patients even if CD38(+) or unmutated had a longer PFS. Furthermore, ZAP-70 positivity was associated with a shorter PFS both within normal karyotype (P < .001) and within the poor-risk cytogenetic subset (P = .02). The predictive value of ZAP-70 expression was confirmed in multivariate analysis. Thus, ZAP-70 protein determined by flow cytometry improves the prognostic significance of cytogenetics and appears to be a better predictor of outcomes than IgV(H) gene mutational status. On this line, we recommend and are also interested in conducting a prospective randomized trial of early intervention versus observation for ZAP-70(+) patients.

ADP-ribosyl Cyclase 1↗
PubMed2007

Comparative analysis of flow cytometric techniques in assessment of ZAP-70 expression in relation to IgVH mutational status in chronic lymphocytic leukemia.

We compared 1 subjective and 5 objective flow cytometric methods to evaluate zeta-associated protein (ZAP-70) expression in relation to immunoglobulin heavy-chain variable-region (IgVH) gene mutational status in 154 samples from 125 patients with chronic lymphocytic leukemia (CLL). ZAP-70 expression determined by all methods used correlated with IgVH gene mutational status, but none of them demonstrated high concordance rates. Of the objective methods, ZAP-70 staining determined as a ratio of molecules of equivalent soluble fluorochrome intensity in CLL cells to that in normal B cells (ZAP-70+ staining in IgVH germline cases, 59%; ZAP-70- in IgVH mutated cases, 75%) or T cells (ZAP-70+ in IgVH germline cases, 66%; ZAP-70- in IgVH mutated cases, 57%) provides the best combination for assigning ZAP-70+ status to IgVH germline and ZAP-70- status to IgVH mutated cases. The subjective method based on ZAP-70 expression in natural killer/T cells gave a similar result, but reproducibility between laboratories may be difficult. Further studies on ZAP-70 expression in relation to clinical parameters may address whether ZAP-70 is an independent prognostic marker for CLL.

DNA Mutational Analysis↗
PubMed1998

Increased Zap-70 association with CD3zeta in CD4 T cells from old mice.

Aging diminishes the amount of phosphotyrosine in the CD3zeta chains of resting and activated mouse CD4 T cells by about threefold and might therefore be expected to a corresponding decline in Zap-70 association with CD3zeta and in Zap-70 kinase function in CD3zeta complexes. We show here that aging leads, unexpectedly, to an approximately twofold increase in the amount of Zap-70 associated with CD3zeta in resting CD4 T cells. There is, however, no effect of age on total intracellular Zap-70 content. Cross-linking CD3 to CD4 leads to an increase of only 50% in the functional activity of Zap-70 in CD3zeta complexes from freshly isolated CD4 T cells of young donors. Compared to Jurkat and HT-2 cells, fresh T cells show both higher baseline levels and lower induced levels of Zap-70 function in CD3zeta complexes. CD4 T cells from old mice have baseline levels of Zap-70 activity similar to those seen in activated T cells from young mice, and these levels do not increase after CD3/CD4 cross-linking. Tyrosine-specific phosphorylation of Zap-70 is also higher at rest in old T cells than in young T cells and inducible only in cells from young donors. These data suggest that age-related defects in T cell activation are not likely to be attributable simply to a decline in Zap-70 association with CD3zeta or to diminished Zap-70 phosphorylation. The increase with age in CD3zeta-Zap association, despite the loss with age in CD3zeta tyrosine phosphorylation, suggests that the pattern of tyrosine phosphate groups among CD3zeta ITAM groups may be different in T cells from young and old donors or that access to ITAM regions within CD3zeta may be blocked by inter- or intramolecular steric hindrance in young CD4 T cells.

Aging↗
PubMed2006

Feasibility of an easily applicable method of ZAP-70 measurement in chronic lymphocytic leukemia in the routine flow cytometry setting: A methodological approach.

Zeta-associated protein 70 (ZAP-70), determined by flow cytometry, has been advocated a surrogate marker of immunoglobulin (Ig)V(H) unmutated status in B chronic lymphocytic leukemia (CLL). The aim of the current study was to test the applicability of an easy flow cytometry protocol for ZAP-70 measurement in CLL samples. Samples from 61 CLL patients and 44 normal subjects were analyzed using a commercial ZAP-70 monoclonal antibody (1E7.2 clone) conjugated with phycoerythrin (PE) and Alexa 488 fluorochromes. Modifications of the published methods led to the structure of a simplified in-house method of ZAP-70 measurement. A three-color approach was used with CD19, CD3 gating comparing with the isotype control provided by the same manufacturer. The cutoff levels for ZAP-70 positivity were defined from a receiver operator characteristic curve in relation to the IgV(H) mutational status and from the ln normalized mean value +2 SD of normal controls. Using the 20% cutoff value for ZAP-70 positivity in CLL patients defined by the literature, ZAP-PE had a sensitivity of 55% and a specificity of 98% in predicting the IgV(H) mutational status, whereas the corresponding values for ZAP-Alexa were 55% and 84%, respectively. Using the 7% cutoff value for CD38 positivity, the sensitivity was 55%, whereas the specificity was 76%. ZAP-70-positive patients showed a shorter time to disease progression in comparison with ZAP-70-negative patients (p < 0.001). In conclusion, the 100% specific prediction of mutational status is accompanied by reduced sensitivity, thus limiting ZAP-70's applicability either as a single marker or combined with CD38 for the assessment of the mutational status of CLL.

ADP-ribosyl Cyclase 1↗
PubMed1996

Mechanism of activation for Zap-70 catalytic activity.

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a zeta-associated protein of M(r) 70,000 (Zap-70) as a critical protein tyrosine kinase in T-cell activation and development. During T-cell activation, Zap-70 becomes associated via its src homology type 2 (SH2) domains with tyrosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) sequences in the cytoplasmic zeta chain of the T-cell receptor. An intriguing conundrum is how Zap-70 is catalytically activated for downstream phosphorylation events. To address this question, we have used purified Zap-70, tyrosine phosphorylated glutathione S-transferase (GST)-Zeta, and GST-Zeta-1 cytoplasmic domains, and various forms of ITAM-containing peptides to see what effect binding of zeta had upon Zap-70 tyrosine kinase activity. The catalytic activity of Zap-70 with respect to autophosphorylation increased approximately 5-fold in the presence of 125 nM phosphorylated GST-Zeta or GST-Zeta-1 cytoplasmic domain. A 20-fold activity increase was observed for phosphorylation of an exogenous substrate. Both activity increases showed a GST-Zeta concentration dependence. The increase in activity was not produced with nonphosphorylated GST-Zeta, phosphorylated zeta, or phosphorylated ITAM-containing peptides. The increase in Zap-70 activity was SH2 mediated and was inhibited by phenylphosphate, Zap-70 SH2, and an antibody specific for Zap-70 SH2 domains. Since GST-Zeta and GST-Zeta-1 exist as dimers, the data suggest Zap-70 is activated upon binding a dimeric form of phosphorylated zeta and not by peptide fragments containing a single phosphorylated ITAM. Taken together, these data indicate that the catalytic activity of Zap-70 is most likely activated by a trans-phosphorylation mechanism.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of human ZAP-70 protein-tyrosine kinase from a baculovirus expression system.

The ZAP-70 protein tyrosine kinase is essential for T cell antigen receptor (TCR)-mediated signaling. The absence of ZAP-70 results in impaired differentiation of T cells and a lack of responsiveness to antigenic stimulation. In order to study the characteristics of ZAP-70 in vitro, we overexpressed an epitopically tagged human ZAP-70 in a recombinant baculovirus expression system and purified it by column chromatography. The kinase activity of purified, recombinant ZAP-70 required cation and exhibited a strong preference for Mn2+ over Mg2+. The apparent Km of ZAP-70 for ATP was approximately 3.0 microM. The activity of the recombinant ZAP-70, unlike that of the homologous protein tyrosine kinase, Syk, was not affected by binding of TCR-derived tyrosine phosphorylated immunoreceptor tyrosine-based activation motif peptides. Several proteins were tested as potential in vitro substrates of ZAP-70. Only alpha-tubulin and the cytoplasmic fragment of human erythrocyte band 3 (cfb3), which have a region of sequence identity at the phosphorylation site, proved to be good substrates, exhibiting Kmvalues of approximately 3.3 and approximately 2.5 microM, respectively ([ATP] = 50 microM). alpha- and beta-Casein were poor substrates for ZAP-70, and no activity toward enolase, myelin basic protein, calmodulin, histone proteins, or angiotensin could be detected. In contrast to the T cell protein tyrosine kinase, Lck, ZAP-70 did not phosphorylate the cytoplasmic portion of the TCRzeta chain or short peptides corresponding to the CD3epsilon or the TCRzeta immunoreceptor tyrosine-based activation motifs. Our studies suggest that ZAP-70 exhibits a high degree of substrate specificity.

Amino Acid Sequence↗
PubMed2004

Expression of ZAP-70 protein correlates with disease stage in chronic lymphocytic leukemia and is associated with, but not generally restricted to, non-mutated Ig VH status.

The mutational status of immunoglobulin variable region genes (Ig VH) is a well established prognostic parameter in chronic lymphocytic leukemia (CLL). Recently, a subset of genes with a characteristic expression profile correlating with the mutational status of B-CLLs has been identified. One of the overexpressed genes in the prognostically unfavorable group of CLL patients with unmutated Ig VH genes encodes for the protein tyrosine kinase ZAP-70, which is physiologically involved in T-cell signaling. Since ZAP-70 has been described to be prognostically relevant in CLL, we analyzed the possible relationship of its expression to the mutational status of Ig VH genes as well as to other prognostic factors in CLL and indolent lymphomas. The mutational status of Ig VH genes was analyzed by seminested PCR, direct sequencing and comparison with the sequences of the EMBL databases in 60 samples of patients with B-CLL and 18 samples of patients with indolent B-cell malignancies. ZAP-70 protein expression was assessed in all samples by immunoblotting and for semiquantitative analysis the ratio of ZAP-70 to tubulin expression was calculated. ZAP-70 protein was found to be expressed in all investigated B-cell malignancies. Expression levels varied within a wide range in each entity. The highest mean level of ZAP-70 expression was observed in unmutated B-CLLs, however, with broad expression variability. High levels of ZAP-70 expression correlated with higher stage Binet B or C and with unmutated Ig VH genes. Overall survival rates estimated by Kaplan-Meier curves did not differ among patients with high or low ZAP-70 expression. We conclude that ZAP-70 is associated with the mutational status of Ig VH genes, but this expression pattern is not present in all individual cases. Furthermore, high levels of ZAP-70 correlated with Binet stages B or C indicating an involvement of ZAP-70 in mechanisms promoting growth of B-CLL cells.

Adult↗
PubMed1995

Binding of ZAP-70 to phosphorylated T-cell receptor zeta and eta enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins.

ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.

Amino Acid Sequence↗
PubMed1998

Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.

T-cell antigen receptor (TCR) engagement activates multiple protein tyrosine kinases (PTKs), including the Src family member, Lck, and the Syk-related PTK, ZAP-70. Studies in ZAP-70-deficient humans have demonstrated that ZAP-70 plays crucial roles in T-cell activation and development. However, progress toward a detailed understanding of the regulation and function of ZAP-70 during TCR signaling has been hampered by the lack of a suitable T-cell model for biochemical and genetic analyses. In this report, we describe the isolation and phenotypic characterization of a Syk- and ZAP-70-negative somatic mutant derived from the Jurkat T-cell line. The P116 cell line displays severe defects in TCR-induced signaling functions, including protein tyrosine phosphorylation, intracellular Ca2+ mobilization, and interleukin-2 promoter-driven transcription. These signaling defects were fully reversed by reintroduction of catalytically active versions of either Syk or ZAP-70 into the P116 cells. However, in contrast to ZAP-70 expression, Syk expression triggered a significant degree of cellular activation in the absence of TCR ligation. Transfection experiments with ZAP-70-Syk chimeric proteins indicated that both the amino-terminal regulatory regions and the carboxy-terminal catalytic domains of Syk and ZAP-70 contribute to the distinctive functional properties of these PTKs. These studies underscore the crucial role of ZAP-70 in TCR signaling and offer a powerful genetic model for further analyses of ZAP-70 regulation and function in T cells.

Clone Cells↗
PubMed2002

Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia.

We examined isolated leukemia B cells of patients with chronic lymphocytic leukemia (CLL) for expression of zeta-associated protein 70 (ZAP-70). CLL B cells that have nonmutated immunoglobulin variable region genes (V genes) expressed levels of ZAP-70 protein that were comparable to those expressed by normal blood T cells. In contrast, CLL B cells that had mutated immunoglobulin variable V genes, or that had low-level expression of CD38, generally did not express detectable amounts of ZAP-70 protein. Leukemia cells from identical twins with CLL were found discordant for expression of ZAP-70, suggesting that B-cell expression of ZAP-70 is not genetically predetermined. Ligation of the B-cell receptor (BCR) complex on CLL cells that expressed ZAP-70 induced significantly greater tyrosine phosphorylation of cytosolic proteins, including p72(Syk), than did similar stimulation of CLL cells that did not express ZAP-70. Also, exceptional cases of CLL cells that expressed mutated immunoglobulin V genes and ZAP-70 also experienced higher levels tyrosine phosphorylation of such cytosolic proteins following BCR ligation. Following BCR ligation, ZAP-70 underwent tyrosine phosphorylation and became associated with surface immunoglobulin and CD79b, arguing for the involvement of ZAP-70 in BCR signaling. These data indicate that expression of ZAP-70 is associated with enhanced signal transduction via the BCR complex, which may contribute to the more aggressive clinical course associated with CLL cells that express nonmutated immunoglobulin receptors.

B-Lymphocytes↗
PubMed2000

The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.

The protooncogene product Cbl has emerged as a negative regulator of tyrosine kinases. We have shown previously that Cbl binds to ZAP-70 through its N-terminal tyrosine kinase binding (TKB) domain. In this study, we demonstrate that overexpression of Cbl in Jurkat T cells decreases the TCR-induced phosphorylation of ZAP-70 and other cellular phosphoproteins. Coexpression of Cbl with ZAP-70 in COS cells reproduced the Cbl-induced reduction in the level of phosphorylated ZAP-70. The effect of Cbl was eliminated by the TKB-inactivating G306E mutation in Cbl as well as by a phenylalanine mutation of Tyr292 within the TKB domain binding site on ZAP-70. Notably, the oncogenic Cbl-70Z/3 mutant associated with ZAP-70, but did not reduce the levels of phosphorylated ZAP-70. Overexpression of Cbl, but not Cbl-G306E, in Jurkat T cells led to a decrease in the TCR-induced NF-AT luciferase reporter activity. Overexpression of the TKB domain itself, but not its G306E mutant, functioned in a dominant-negative manner and led to an increase in NF-AT reporter activity. Cbl-70Z/3-overexpressing cells exhibited an increase in both basal and TCR-induced NF-AT luciferase reporter activity, and this trend was reversed by the G306E mutation. Finally, by reconstituting a ZAP-70-deficient Jurkat T cell line, p116, we demonstrate that wild-type ZAP-70 is susceptible to the negative regulatory effect of Cbl, whereas the ZAP-70-Y292F mutant is resistant. Together, our results establish that the linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.

Animals↗
PubMed2005

ZAP-70 tyrosine kinase is constitutively expressed and phosphorylated in B-lineage acute lymphoblastic leukemia cells.

BACKGROUND AND OBJECTIVES: Zeta-associated protein 70 (ZAP-70), a member of the Syk family of protein tyrosine kinases, is normally expressed in T and NK cells. While little is known about ZAP-70 expression in normal human B cells, it has been reported that ZAP-70 is expressed in a subset of patients with chronic lymphocytic leukemia (CLL) with a poor prognosis. In this study, we examined the expression and phosphorylation status of ZAP-70 in B-lineage acute lymphoblastic leukemia (Blin-ALL). DESIGN AND METHODS: First, ZAP-70 protein expression was assessed by Western blotting and flow cytometry and ZAP-70 mRNA transcripts were analyzed by reverse transcription polymerase chain reaction (RT-PCR) on human precursor B cell lines. Experiments were then carried out on cells obtained from 18 patients with Blin-ALL and from normal human bone marrow. RESULTS: ZAP-70 was constitutively expressed and phosphorylated on tyr319 in human precursor Blin-ALL cell lines as well as in primary B leukemic cells from all examined Blin-ALL patients with pro-B, pre-B and B phenotypes, but not in malignant myeloid cells. Importantly, analysis of normal human bone marrow revealed expression of ZAP-70 transcripts only in the CD34+ cell fraction (either CD19-CD10- or CD19+CD10+) but not in the CD34- cell fraction (CD19+sIgM- pre-B cells or CD19+sIgM+ immature B cells). INTERPRETATION AND CONCLUSIONS: ZAP-70 was found to be expressed in the CD34+ normal bone marrow compartment including earlier B-cell progenitors, but not in CD34- pre-B and immature B cells. By contrast, ZAP-70 was consistently expressed and phosphorylated in Blin-ALL cells. Further studies are required to determine whether ZAP-70 may play a pathophysiological role in Blin-ALL.

Adult↗
PubMed2006

Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome.

BACKGROUND: ZAP-70 has emerged as a potential pivotal prognostic marker for patients with chronic lymphocytic leukemia (CLL), which could replace immunoglobulin heavy chain mutation status. Although several flow cytometry assays have been described for assessing ZAP-70 in CLL, certain technical and scientific issues remain unsolved, which have prevented results of this crucial test from being reported, even in the best routine flow cytometry laboratories. In this report, we aimed to solve some of these issues by providing a computerized quantitative flow cytometric assay for ZAP-70 within the entire CLL population, which would be easy to perform and enable standardization between laboratories. METHODS: Intracellular ZAP-70 levels in CLL and normal B cells were assessed by molecules of equivalent soluble fluorochrome (MESF), employing Quantum FITC MESF calibration beads to establish a standard curve relating channel value to fluorescence intensity in MESF units and the QuickCal v. 2.2 program (www.bangslabs.com) and clinical relevance of the data was determined. RESULTS: The average ZAP-70 expression value in the CD19(+)/CD5(+) cells from 35 CLL patients was 103,701 MESF when compared with 12,621 MESF in B cells from 20 normal blood samples. "Low" and "high" ZAP-70 CLL subgroups were defined. Patients with "high ZAP-70 MESF" CLL had a shorter time to disease progression (P = 0.0005) and a more advanced clinical stage (P = 0.0018) when compared with patients in the "low ZAP-70 MESF" CLL subgroup. CONCLUSIONS: This quantitative analysis method can be employed to obtain a more specific and highly accurate assessment of ZAP-70 levels in CLL cells. The method can easily be standardized, in any routine flow laboratory, thereby improving reproducibility and reliability of ZAP-70 analysis.

Aged↗
PubMed2006

A robust ratio metric method for analysis of Zap-70 expression in chronic lymphocytic leukemia (CLL).

Since Zap-70 expression in chronic lymphocytic leukemia (CLL) cells correlates with a lack of somatic mutation of the immunoglobulin variable heavy chain (IgVH) genes, it has been proposed as a surrogate marker for disease prognosis. However, published studies of Zap-70 expression have used different commercial antibodies and analytic strategies. This study was undertaken to determine if any strategy was broadly applicable in a clinical flow cytometry laboratory. Expression of Zap-70 was determined in 37 CLL patients using four different commercial antibodies. T, NK, and CLL cells were identified by immunophenotyping along with Zap-70 expression. Data was analyzed in terms of both percent of CLL cells expressing Zap-70 and the ratio of Zap-70 expression in CLL cells compared to that in T + NK cells. Three Zap-70 antibodies showed wide ranges of Zap-70 expression as a percentage of tumor cells, while a fourth gave consistently elevated results. Comparing the percent Zap-70 expression with any two antibodies gave poor correlations (r(2) = 0.45-0.63). Our results indicated that the previous analytical strategies were not reproducible. A ratio metric is proposed, which gave better correlations (r(2) as high as 0.95) and would allow separation of CLL patients with elevated or decreased Zap-70 expression.

Antibodies, Monoclonal↗
PubMed2006

Discordant results of flow cytometric ZAP-70 expression status in B-CLL samples if different gating strategies are applied.

BACKGROUND: Recent studies have identified ZAP-70 expression status as an excellent prognostic parameter in chronic lymphocytic leukemia (CLL). ZAP-70 expression can be determined by direct antibody staining followed by flow cytometric analysis. However, there are currently several different gating strategies for analysis. We compared those strategies for ZAP-70 expression analysis. METHODS: One hundred and one patients with B-CLL were analyzed employing a directly labeled alexa-fluor-488-ZAP-70-antibody. In 27 cases, we additionally measured and analyzed ZAP-70 expression, together with healthy controls as described previously. RESULTS: Applying either T-/NK-cell isotype or healthy control analysis strategies on patient samples that were processed in parallel revealed discrepant results in 48% (12/25) of all cases. Taken together with the 74 B-CLL patients, who were analyzed with regard to average reference values, disconcordant results were obtained in 58% of the samples. We demonstrate that high variances in ZAP-70 T-/NK-cell staining within B-CLL patients, paired with a close proximity of ZAP-70 B-cell values to the suggested cut-off levels, may lead to interpretation difficulties of ZAP-70 status. CONCLUSIONS: We conclude that different gating strategies for determining flow cytometric ZAP-70 expression status produce highly discordant results. Further standardization is required before ZAP-70 can be used as a reliable prognostic parameter in immunophenotyping of B-CLL.

Adult↗