Analysis Of ZAP-70 Expression Research Articles

Validation of ZAP-70 methylation and its relative significance in predicting outcome in chronic lymphocytic leukemia

AbstractZAP-70 methylation 223 nucleotides downstream of transcription start (CpG+223) predicts outcome in chronic lymphocytic leukemia (CLL), but its impact relative to CD38 and ZAP-70 expression or immunoglobulin heavy chain variable region (IGHV) status is uncertain. Additionally, standardizing ZAP-70 expression analysis has been unsuccessful. CpG+223 methylation was quantitatively determined in 295 untreated CLL cases using MassARRAY. Impact on clinical outcome vs CD38 and ZAP-70 expression and IGHV status was evaluated. Cases with low methylation (<20%) had significantly shortened time to first treatment (TT) and overall survival (OS) (P < .0001). For TT, low methylation defined a large subset of ZAP-70 protein-negative cases with significantly shortened TT (median, 8.0 vs 3.9 years for high vs low methylation; hazard ratio [HR] = 0.43; 95% confidence interval [CI], 0.25-0.74). Conversely, 16 ZAP-70 protein-positive cases with high methylation had poor outcome (median, 1.1 vs 2.3 years for high vs low methylation; HR = 1.62; 95% CI, 0.87-3.03). For OS, ZAP-70 methylation was the strongest risk factor; CD38 and ZAP-70 expression or IGHV status did not significantly improve OS prediction. A pyrosequencing assay was established that reproduced the MassARRAY data (κ coefficient > 0.90). Thus, ZAP-70 CpG+223 methylation represents a superior biomarker for TT and OS that can be feasibly measured, supporting its use in risk-stratifying CLL.

Analysis of ZAP70 expression in adult acute lymphoblastic leukaemia by real time quantitative PCR

BackgroundZAP70 gene expression is associated with poor prognosis in B-cell lymphoproliferative disorders especially chronic lymphocytic leukaemia (CLL) but its role in adult B-ALL has not been established. On diagnostic samples from 76 patients with adult ALL (65 with B-ALL and 11 with T-ALL) ZAP70 mRNA expression levels were studied by real time-quantitative PCR (RT-qPCR) analysis.FindingsA broad distribution of ZAP70 expression was observed in ALL, ranging from 0.002 to 5.3 fold that of the ZAP70 positive Jurkat reference cell line. No association was observed between expression levels and the presence of specific cytogenetic abnormalities. Five cases, including one case of T-ALL, had ZAP70 expression above the level of the Jurkat reference cell line.ConclusionsOur results confirm the frequent expression of ZAP70 in adult ALL. Limited comparisons made did highlight poor-risk patients with high ZAP70 expression, but due to lack of clinical information on patient samples we were unable to directly assess the impact on disease prognosis. ZAP-70 may be an important laboratory assay in adult ALL and further studies are warranted to study a potential correlation with cytogenetic and other genetic markers.

Detection methods of ZAP-70 in chronic lymphocytic leukemia

The clinical course of patients with chronic lymphocytic leukemia (CLL) is highly heterogeneous, with some patients experiencing rapid disease progression and others living for decades without requiring treatment. Immunoglobulin heavy chain variable region (IGHV) mutation status is a powerful prognostic factor in patients with CLL. The presence or absence of IGHV mutation status is currently the gold-standard prognostic factor, but this technique is labor-intensive and costly. The expression of ζ-chain-associated protein kinase 70kDa (ZAP-70), detected by flow cytometry, has been served as a potential surrogate for the absence of IGHV mutation status since 2003. Given the current problems with the standardization of ZAP-70 assessment by flow cytometry, this chapter reviews the analysis of ZAP-70 expression by flow cytometry and the relative experimental conditions using the associated publications for "ZAP-70" and "detection method" on Medline since 2003. Thus, mean fluorescence intensity (MFI) ratio method yielded a more reproducible and easily adaptable method to routine use in the clinical diagnostic laboratory. In conclusion, though ZAP-70 is a valuable prognostic factor in CLL, detection methods of ZAP-70 remain to be fully standardized.

Improved ZAP-70 assay using two clones, multiple methods of analysis and clinical correlation.

In a companion methodological study, we compared two anti-ZAP-70 clones (1E7.2 AF 488 and SBZAP PE) and four selected methods of analysis. Clinical correlations are required for validation. Multicolor flow-cytometric evaluation of ZAP-70, CD38, CD69, CD26, CD49d, and CD27 was tested in 45 untreated-CLL patients. Four methods of ZAP-70 expression analysis and a scoring system were designed. A correlation analysis between ZAP-70 score, immunoglobulin heavy chain variable (IGHV) mutational status, fluorescence in situ hybridization, and these biomarkers was undertaken. There is a strong correlation between ZAP-70 expression and IGHV mutational status. The scoring system for a single reagent (P = 0.0006 or 0.0002) favors the use of multiple methods of analysis. The combined score was substantially equivalent (P = 0.0003). There was also a correlation with del 13q14 (P = 0.017) and trisomy12 (P = 0.011). A correlation for CD38 and ZAP-70 score was seen using both 1E7.2 AF488 and SBZAP PE when ≥20% or ≥7% cutoff was used. A positive correlation was seen for CD49d expression using both reagents. CD26 showed a correlation with ZAP-70 expression, but it was dependent upon the method of analysis. CD69 and CD27 showed no statistically significant correlation. In our study population, ZAP-70 expression is the better predictor of the IGHV mutational status. The correlation analysis confirms that the use of four methods of analysis with a single reagent or both reagents is superior to the use of a single method of analysis. The routine use of CD38, CD49d, and CD26 will require standardization.

Immunohistochemical analysis of ZAP-70 expression in chronic lymphocytic leukemia

This paper shows a protocol for the detection of ZAP-70 expression in B-CLL (B cell chronic lymphocytic leukemia) tumor cells by common immunohistochemical methods. The study was conducted on bone marrow trephine biopsies from 62 B-CLL patients at the time of diagnosis. Immunohistochemical reactions based on peroxidase and alkaline phosphatase reactions were used, as well as double immunofluorescent labeling for ZAP-70 detection as an indirect marker of mutated and unmutated CLL. Clinical relevance of the ZAP-70 expression detection method was assessed using chi2 test between ZAP-70 positivity data and other known prognostic factors, i.e., clinical and cytogenetics data. ZAP-70 was detected in 13 out of 62 patients. Statistically significant results were obtained for ZAP-70 positive cases and known indicators of worse prognosis. Immunohistochemical analysis supported by double immunofluorescent labeling, as shown here, is an easy and reliable technique for the detection of ZAP-70 expression in B-CLL tumor cells applicable in every hematopathology laboratory.

A Scoring System Based on Clinical, Blood Count and Immunophenotypic Data Can Predict the Risk of Disease Progression in Patients with Chronic Lymphocytic Leukemia

There is limited access to advanced molecular and immunophenotypic techniques such as immunoglobulin heavy chain variable mutational status and ZAP-70 expression analysis in many parts of the world. To facilitate the development of a clinical model based on readily available clinical and well-standardized pathological data, we analyzed the well known prognostic factors in chronic lymphocytic leukemia (CLL) and proposed a scoring system. We searched the CLL database and among 465 patients (pts) we included 128 pts where the staging, blood counts, immunophenotypic and follow up data are complete. The baseline characteristics of this group and the significance of the proposed factors on the progression-free survival on univariate analysis are summarized in the table. Progression–free survival (PFS) was defined as the time from diagnosis to the time where treatment for CLL is indicated and if no treatment id indicated, the time of last follow up. In this 5 point scoring system we added one point for the presence of each one of the following factors: male gender, Rai stage 1 or more, lymphocyte count of 20 ×109/l or higher, lymphocyte doubling time less than 12 m, CD 38 expression of 20% or more. We further lumped these groups into 3 groups: no risk factors, one risk factor and tow or more risk factors. Thirty eight patients (30%) were found to have no risk factors and their 5 and 10 year PFS was 100% and 100% respectively. Forty nine pts (38%) have at least one risk factor and their 5 and 10 y PFS was 92% and 81% respectively. Forty pts (31%) have two or more risk factors and their 5 and 10 y PFS was 48% and 20% respectively. Those differences in PFS were significant (p<0.00001). We conclude that readily available clinical and laboratory information in patients with CLL can be utilized to predict the risk of disease progression. Pts with no risk factors can be reliably reassured and do not require regular monitoring. Correlation of these findings with other molecular prognostic factors is needed.Table: Risk factors for disease progressionParameterN (%)5-y PFS10-y PFSp value (UVA)*Sex: Male71 (56)67%43%0.01Female56 (44)91%73%Rai stage: 0103 (80)87%77%<0.00011,2,3,425 (20)49%18%Lymphocyte count: < 20×109/l108 (84)85%60%<0.0001≥20×109/l20 (16)40%40%Lymphocyte doubling time ≥ 12m111 (87)84%68%0.001<12 m17 (13)50%22%CD38 <20%99 (77)83%67%0.006≥20%29 (23)62%36%*Univariate analysis [Display omitted]

Analysis of ZAP-70 Expression in Chronic Lymphocytic Leukemia (CLL) Using Kolmogorov-Smirnov (KS) Statistics.

ZAP-70 is a prognostic marker in CLL and has been shown to correlate closely with VH mutational status in most studies. Assays that require the setting of markers using either T-cells, or a matched isotype control to determine ZAP-70 expression in CLL are subjective and prone to analytical and inter-operator variability. We examined the use of the KS statistical test to analyze histogram overlays as an objective approach to the assessment of ZAP-70 expression in 18 CLL patients with known VH mutational status (VH mutated; n=11, VH unmutated; n=7). Use of the KS statistical test to compare ZAP-70 expression of T cells with B cells in CLL has been recently described by Van Bockstaele et al.(Cytometry Part B 70B:302-308. 2006). In contrast to this approach we used a B cell gating assay with a pre-conjugated alexa-fluor 488 ZAP-70 antibody clone 1E7.2 and an alexa-fluor 488 matched isotype control manufactured by Caltag Laboratories (Burlingame, CA) as previously described by our group. Modifications were made to allow the determination of KS ‘D'values. Statistical analysis of our resulting KS data using a t-test demonstrated a significant difference in ZAP-70 KS ‘D' values between the two VH mutational groups (p=0.0001). All patients with a KS ‘D' value of 0.5 or below were VH mutated, whilst all patients with a KS ‘D' value of 0.7 or above were VH unmutated, with some cross-over between these values. At a threshold KS ‘D' value of 0.6, 10 of the 11 VH mutated patients were correctly classified (1 patient had a ‘D' value of 0.68), whilst 6 of the 7 VH unmutated patients were correctly classified (1 patient had a ‘D' value of 0.59) The sensitivity and specificity of using a threshold KS ‘D' value of 0.6 was 86% and 91% respectively. Analysis of ZAP-70 expression using the KS statistic is operator independent and thus provides a means for a more reproducible analytical approach in the laboratory. We acknowledge that our initial sample size was small and requires further investigation using a larger sample size to fully validate the diagnostic value of using KS ‘D' values to determine ZAP-70 expression in CLL.

An optimized whole blood method for flow cytometric measurement of ZAP‐70 protein expression in chronic lymphocytic leukemia

ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations. A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4-17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-ZAP-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens. Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients. The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory.

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.

Kolmogorov–Smirnov statistical test for analysis of ZAP‐70 expression in B‐CLL, compared with quantitative PCR and IgVH mutation status

ZAP-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgV(H)) mutation status, which is known as a prognostic marker in B-cell chronic lymphocytic leukemia (CLL). The flow cytometric analysis of ZAP-70 suffers from difficulties in standardization and interpretation. We applied the Kolmogorov-Smirnov (KS) statistical test to make analysis more straightforward. We examined ZAP-70 expression by flow cytometry in 53 patients with CLL. Analysis was performed as initially described by Crespo et al. (New England J Med 2003; 348:1764-1775) and alternatively by application of the KS statistical test comparing T cells with B cells. Receiver-operating-characteristics (ROC)-curve analyses were performed to determine the optimal cut-off values for ZAP-70 measured by the two approaches. ZAP-70 protein expression was compared with ZAP-70 mRNA expression measured by a quantitative PCR (qPCR) and with the IgV(H) mutation status. Both flow cytometric analyses correlated well with the molecular technique and proved to be of equal value in predicting the IgV(H) mutation status. Applying the KS test is reproducible, simple, straightforward, and overcomes a number of difficulties encountered in the Crespo-method. The KS statistical test is an essential part of the software delivered with modern routine analytical flow cytometers and is well suited for analysis of ZAP-70 expression in CLL.

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

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. 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. 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. 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.

Analysis with the LightCycler System® Identifies a Highly Significant Correlation between ZAP-70 mRNA Expression and Immunoglobulin Variable Heavy Chain Gene Mutational Status in Chronic Lymphocytic Leukemia.

The presence or absence of somatic mutations in the immunoglobulin variable heavy chain (IgVH) region of chronic lymphocytic leukemia (CLL) B cells has prognostic information already at the time of diagnosis. The lack of somatic mutations in about 50% of the CLL patients significantly separates those with poor prognosis from those with a more indolent course that carry somatic mutations in their IgVH gene. However, the analysis of this marker is time consuming. Global gene expression analysis of CLL cells identified the expression of the T cells marker zeta chain associated protein (ZAP-70) as a surrogate marker for an unmutated IgVH. In order to analyze ZAP-70 expression in CLL cells using a relative quantification approach, we developed an assay**For research use only. Not available in distribution. on the LightCycler® instrument. Here, we analyzed a cohort of 100 samples for ZAP-70 mRNA and compared the results with the IgVH mutational status. Mononuclear cells were isolated from blood (n=80) or from bone marrow (n=20). To exclude ZAP-70-expressing T and natural killer cells from analysis, cells were enriched for B cells to a purity of > 99% by CD19 magnetic beads positive selection and mRNA was isolated using a MagNA Pure LC instrument. cDNA was reverse transcribed from an equivalent of 5x 106 cells and used for the analysis of the IgVH status as described (Blood . 2003; 101:2049–2053) as well as for the analysis of ZAP-70 expression with a LightCycler® instrument. ZAP-70 expression levels as the target gene were normalized to the houskeeping gene ABL using relative quantitation. mRNA of the Jurkat cell line was used as a calibrator. In preliminary experiments, ABL was selected from a total of 4 different housekeeping genes based on its expression levels, which were in the range of ZAP-70 expression, and based on the resulting correlation to the IgVH status. Thus, results for ZAP-70 gene expression are given as normalized ratio. ZAP-70 mRNA expression (n=100) revealed a highly significant correlation to an unmutated IgVH and low ZAP-70 expression correlated to a mutated IgVH (r2=0,692, Spearman). A cut off of 0.5 ZAP-70 expression was optimal to distinguish samples with mutated from unmutated IgVH, resulting in 80 concordant samples (80%). Of the 11 mutated IgVHs with discordant ZAP-70 expression 7 (64%) had borderline mutation status and 4 (36%) expressed the poor prognosis VH3-21 gene. 7(78%) of the 9 unmutated IgVHs with low ZAP-70 expression were characterized by adverse cytogenetic features. In summary, the results of the present study show that the LightCycler® system based quantitative RT-PCR is suitable to achieve a highly significant correlation between the IgVH status and ZAP-70 expression in CLL samples. Unlike the flow cytometric detection of ZAP-70 protein expression, which uses ZAP-70 of endogenous T cells as an internal control, the assay* developed by us might be easier to standardize between laboratories and therefore, might be applicable for future routine applications.

FCR-3 as Frontline Therapy for Patients with Chronic Lymphocytic Leukemia (CLL).

The combination regimen of Fludarabine, Cyclophosphamide, and Rituximab (FCR) has produced high overall response (OR) rates and complete remission (CR) rates in patients receiving this as initial treatment for CLL. Earlier studies have shown that dose-dense or dose-intensified regimens of single-agent Rituximab produced higher response rates in patients with CLL. The current regimen, FCR-3, was based on the hypothesis that increasing the amount of Rituximab in the combination might improve response rates and remission duration. Doses of chemotherapy were Fludarabine 25 mg/m2/Dx3, and Cyclophosphamide 250 mg/m2/Dx3 given monthly. Rituximab was given at 375 mg/m2 as the first dose and 500 mg/m2 for all subsequent doses. On each day of the chemotherapy a dose of Rituximab was given so 3 doses were given monthly. With the previous FCR regimen 45% of patients achieved a CR or nPR and had <5% CD19+ 5 positive cells in the marrow at the completion of 3 cycles of therapy (rapid response, RR). The median time to progression (TTP) in that group has not been reached; it was 3 years in patients who did not achieve that endpoint (p<.001). The current protocol aimed to increase that response rate from 45% to 60%. Sixty-five patients were treated. Eighty percent were men. The median age was 59 years (27–82). Rai Stage 3–4 disease was present in 25% of patients. Median WBC count was 92.4 x 103/ul (7.9–363). Median B2-microglobulin was 3.8 (1.6–10.1). Unfavorable FISH abnormalities were present in 35% of 52 evaluable patients. Somatic hypermutation status was available for 44 patients; 61% were unmutated. ZAP-70 expression analysis performed by immunostaining or flow cytometry was positive in 62% of 47 evaluable patients. Results of FCR-3 in comparison to FCR are shown in the table.3 Cycles6 CyclesNo.RR(%)ORCRFlow<5%FCR30045957282FCR-36545946574PCR negativity using consensus primer PCR was achieved in 49% of patients at the end of therapy. Median number of days between courses ranges from 29–35 per course (overall range 27–95). Eighty-five percent of patients completed 6 cycles. No patient has progressed with a median follow-up of 10 months. With limited follow-up the addition of 3 doses of Rituximab to FC chemotherapy does not appear to provide greater benefit than one dose.

Immunohistochemical analysis of ZAP-70 expression in B-cell lymphoid neoplasms

ZAP-70 is a tyrosine kinase that participates in early B-cell differentiation and is a prognostic factor in chronic lymphocytic leukaemia (CLL), where it is associated with an unmutated configuration of the IgV(H) genes. In this study ZAP-70 expression was studied by immunohistochemistry in a spectrum of B-cell lymphoid neoplasms; this staining method was compared with flow cytometry, and the relationship of ZAP-70 expression to mutational status and prognosis was assessed. 242 tissue samples from 225 patients with B-cell lymphoid neoplasms arising at different maturational stages were included. Flow cytometry was performed in all CLL cases (n = 52). IgV(H) mutational status was determined in 25 CLL and 12 mantle cell lymphoma (MCL) patients. ZAP-70 was positive in 34/52 (65%) CLL, 9/31 (31%) Burkitt's lymphoma, 2/7 (29%) lymphoblastic lymphomas, 3/36 (8%) MCL, 1/23 (4%) marginal zone lymphoma, and 1/45 (2%) diffuse large B-cell lymphomas, but in none of the 19 follicular lymphomas or the 14 Hodgkin lymphomas. An identical ZAP-70 pattern was obtained in six patients with simultaneous biopsies from different sites and in 12 patients with sequential biopsies. Immunohistochemistry and flow cytometry gave identical results in 48 the 52 CLLs. All but one ZAP-70-positive CLL had IgV(H) gene in an unmutated configuration, whereas all but one ZAP-70-negative CLL had somatically hypermutated IgV(H). The 12 MCLs analysed were ZAP-70 negative regardless of IgV(H) mutational status (4 mutated, 8 unmutated). ZAP-70 positive CLL was associated with a shorter overall survival (median time 103 months vs. 293 months, p = 0.01) and a shorter time to disease progression (median time 26 months vs. 60 months, p = 0.01). In conclusion, ZAP-70 is expressed in several types of B-cell neoplasm and is easily detected by immunohistochemistry, providing a useful prognostic marker in patients with CLL from whom no other material is available or when other techniques for its assessment cannot be performed.

Quantitation of Zap-70 Expression in B-CLLCells Employing a Novel Flow Cytometry Analysis Method.

IgVH mutational status and molecular cytogenetics have dramatically improved the ability to predict the prognosis of CLL patients. These tests, however, are highly sophisticated, complex and costly for routine use. ZAP-70, a syk family tyrosine kinase normally expressed in T cells, is a newly described marker which correlates with clinical progression and shorter survival in CLL. A flow cytometry assay to detect ZAP-70 described by Crespo et al (1), appears to be the simplest approach for routine clinical stratification in B-CLL. It is highly informative, and has a strong correlation between the expression of ZAP-70 in CLL cells and clinical outcome. However, in this analysis there are some technical aspects that should be improved to enable it to be standardized as a routine flow cytometry assay. ZAP-70 expression in B-CLL cells is not quantitative but assessed relative to its expression in the T- and NK cells (CD3+, CD56+). This approach can be problematic at times, as ZAP-70 levels in T cells vary in CLL patients as well as in normal controls, probably due to its up regulation following activation. An additional quandary in this assay is that all results are recorded relative to the subjectively delineated T-cell gate. Accordingly, small changes in expression in the T cells can significantly alter the results obtained in some B-CLL samples. In this study we aimed to improve the resolution of the assay by performing a quantitative analysis of ZAP-70 expression within the B-CLL cell population which is uncoupled from T cells. Blood samples were stained by the method described by Crespo et al (1) and ZAP 70 levels in B cell populations in CLL patients (CD19+CD5+) and in healthy volunteers (CD19+) were determined using a standard curve generated by an absolute fluorescent standard of FITC high levels beads with a range of 50–2000 x103 molecules of equivalent soluble fluorochrome (MESF) units per microsphere. Quantitation of expression levels were generated using Quick Cal V2.2 via www.bangslab.com. (Bangs laboratories). Using this analysis system the mean expression levels of ZAP 70 were calculated in healthy B cells (n=11) to be 11,177±1812 MESF units while in CLL (n=36) the mean value was >143,000 MESF units. To determine the reliability of this new method and its clinical relevance we compared our results to data generated using the analysis method of Crespo et al (1). We found a significant correlation between the two methods (r2 = 0.7558). Using ROC curve analysis with maximum sensitivity and specificity, our minimum positive value was found to be 46,700 MESF, with >95% sensitivity at 27,000 MESF and >92% specificity at 67,000 MESF and a Pearson correlation of 0.877 (P<0.0005). We conclude that this assay can provide a more reproducible and reliable analysis of Zap-70 expression in B-CLL, which is easily standardized. This analysis is highly specific as it is quantitative, not subjective and uncoupled from T cell activation in the sample.

Incidence of ZAP70 Positivity in Fresh Samples from Unselected Patients Presenting with a B-Cell Lymphoproliferative Disorder.

The expression of ZAP70 has been shown to strongly correlate with unmutated immunoglobulin VH in chronic lymphocytic leukaemia (CLL) cells, which in turn has been shown to distinguish a CLL subgroup of poor prognosis. Retrospective studies have linked ZAP70 expression to a more aggressive clinical course and poorer overall survival in CLL. Previous studies on ZAP70 have largely utilised frozen material. We wanted to avoid the biases of using stored material, potential artefacts of the freeze/thawing process and expand our study beyond CLL. Hence, we analysed ZAP70 by flow cytometry in 184 consecutive cases of B-cell lymphoproliferative disorder (LPD). Only fresh peripheral blood (PB) samples were used. A combination of ZAP 70 (Upstate Biotechnology), CD3 PE and CD56 PE (Coulter Immunotech) antibodies (Ab) were used to exclude the contaminating effects of T and NK cells. CD3 and CD56 staining was followed by fixation and permeabilisation (Dako Intracellular Antigen Kit) and addition of anti-ZAP70 Ab. Indirect labelling of ZAP70 was performed using a secondary conjugate IgG(H+L)-FITC (Southern Biotechnology). Positivity was established at the 20% threshold. 143 cases of CLL were examined for ZAP70 expression: 47 (33%) were positive, with the level of positivity ranging from 20 to 91% (median 41%). Three cases initially ascribed as negative, were subsequently found to be positive upon retesting at a later date. The ZAP70 negative cases of CLL had a median value of 1% (range, 0 – 14%). 41 non CLL cases were examined for ZAP70 expression, mantle cell lymphoma (MCL, 16 cases), hairy cell leukaemia (HCL, 3), diffuse large B-cell lymphoma (2) and 20 cases of B-cell LPD that could not be categorised further. Of these non CLL cases, only 4 were positive for ZAP 70 expression and all of these were cases of MCL (24 – 39% positivity.) The ZAP70 negative cases of MCL had a median value of 0% (range 0–17%). No other non CLL B-cell LPD showed levels of expression >10%. This study of flow cytometric analysis of ZAP70 expression in fresh, presentation PB of all LPDs referred to our laboratory revealed that 33% of CLL and 25% of MCL cases were positive. ZAP70 expression in CLL is bimodal (clearly positive or negative using the 20% threshold) with very few cases in the 10–19% range. No other B-cell LPDs were positive for ZAP70. As ZAP70 testing is now part of our routine LPD panel, ZAP70 positivity provides both diagnostic and prognostic information in CLL, helping to distinguish difficult CLL cases from non CLL (other than MCL). Our 3 cases of changing status may reflect improvements in our protocol or may indicate that ZAP70 status is variable over the course of the disease.