Comparison of flow cytometric methods for the measurement of ZAP-70 expression in a routine diagnostic laboratory

Chronic lymphocytic leukaemia (CLL) follows a variable clinical course with patient survival ranging from only a few years despite treatment, to several decades in patients who may never require clinical intervention. Determination of the mutational status of a patient's immunoglobulin heavy chain variable region (Ig V(H)) gene has been used to provide prognostic information, but this assay is not available in most laboratories. The discovery of the expression of the protein tyrosine kinase zeta-associated protein (ZAP)-70 in V(H)-unmutated CLL cases led to its proposal as a surrogate marker for V(H) status. This study investigated the measurement of ZAP-70 expression in CLL using different flow cytometric protocols. Two different antibodies and two different staining methods were compared. The Caltag ZAP-70 antibody and Fix & Perm kit were the easiest to use and were the most sensitive and specific combination, with 91% concordance between ZAP-70 expression and V(H) status. Three patients (9%) were discordant (two V(H) mutated/ZAP-70 positive, and one V(H) unmutated/ZAP-70 negative). No correlation existed between CD38 and either ZAP-70 expression or V(H) status. Measurement of ZAP-70 expression using the Caltag antibody/kit combination provides a standardized flow cytometric method that could be introduced into a routine CLL immunophenotyping panel in a clinical diagnostic laboratory.     

ZAP-70 in B-cell chronic lymphocytic leukemia: clinical significance and methods of detection

B-cell chronic lymphocytic leukemia (B-CLL) represents a heterogeneous disease with highly variable prognosis. Clinical staging systems (Rai, Binet) fail to accurately predict the prognosis of individual patients, especially in early stages. Modern prognostic markers, mainly the mutational status of the variable regions of immunoglobulin heavy chains (IgVH) and genetic aberrations, allow more accurate risk stratification. Assessment of the expression of intracellular protein tyrosine kinase ZAP-70 represents not only a potential surrogate marker for the technically difficult and routinely unavailable assessment of the IgVH mutational status, but might also be an independent prognostic factor. Study of ZAP-70 function in B-cells has broadened our knowledge on the pathogenesis of B-CLL. Routine ZAP-70 assessment has been hindered so far mainly by the lack of harmonization and standardization of the available methods of detection.     

Expression level of lipoprotein lipase in Chinese patients with chronic lymphocytic leukemia and its correlation with other prognostic factors

Chronic lymphocytic leukemia (CLL) is the most common adult form of leukemia in the Western world, however, infrequent in the Eastern. It shows a remarkable heterogeneity, with some patients having an almost normal lifespan, others surviving only several years after diagnosis despite intensive therapy. To investigate lipoprotein lipase (LPL) expression level in Chinese patients with CLL and its correlation with other prognostic factors, including immunoglobulin heavy-chain variable region (IgVH) mutation status, Binet stages, ZAP-70 protein and CD38 expression level, semiquantitative RT-PCR was used to detect LPL expression in peripheral blood samples of 58 Chinese patients with CLL. LPL expression level was significantly correlated with IgVH mutational status (r = 0.348, P = 0.010), Binet stages (r = 0.276, P = 0.036), ZAP-70 protein (r = 0.431, P = 0.001) and CD38 (r = 0.546, P < 0.001). Patients with unmutated IgVH genes had higher expression of LPL than patients with IgVH mutations. The higher expression level of LPL was also associated with higher level of ZAP-70 and CD38, and more aggressive Binet stage. We also analyzed LPL expression in different cytogenetic subgroups. Higher LPL level was found in patients with unfavorable cytogenetic aberrations (deletion in 17p13 or 11q22) in contrast to lower level in good risk cytogenetics (deletion in 13q as the sole abnormality) (r = 0.404, P = 0.002). It was showed that LPL expression correlates with IgVH mutational status and other clinical or laboratory prognostic factors, and might be applied for the assessment of prognosis in patients with CLL.     

The effect of IgVH mutational status on the induction of apoptosis by rituximab in patients with heavily pretreated B-cell chronic lymphocytic leukemia: evidence from a clinical phase I/II trial

We investigated tumor cell apoptosis in vivo in 14 heavily pretreated patients with B-cell chronic lymphocytic leukemia undergoing rituximab monotherapy. Apoptosis induction was more pronounced in patients with mutated IgVH genes than in those with unmutated IgVH genes, independently of the levels of expression of CD20, CD38, and ZAP-70 and of the presence of 17p deletion. Our results suggest an association between IgVH gene mutational status and rituximab-induced apoptosis.     

Clinical utility of molecular and flow cytometric markers in chronic lymphocytic leukaemia

Background: Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease. While immunoglobulin variable region heavy chain (IgVH) mutational status remains the ‘gold standard’ in molecular prognostication, a range of additional markers is increasingly being used in clinical trials. As awareness of trial data increases, requests to determine these prognostic markers for new CLL patients are becoming more prevalent in Australia. Aim: To explore the clinical utility of currently available prognostic markers for CLL in an Australian cohort. Methods: IgVH mutational status and gene usage was determined and compared with other reported immunophenotypic markers, cytogenetics and clinical outcome as defined by treatment‐free survival (TFS), lymphocyte doubling time and clinical stage in a cohort of 65 CLL patients. Results: An unmutated IgVH gene, high expression of CD38, ZAP‐70, CD25, CD49d, CD54 or low expression of CD49c was associated with shorter TFS indicating an adverse clinical prognosis in our cohort. High expression of each of CD38, ZAP‐70, CD49d and CD54 was significantly associated with an unmutated IgVH gene; however, associations were not absolute. IgVH and CD25 expression retained their significance in multivariate analysis. Concordant CD25^high/IgVH unmutated CLL patients had the shortest median TFS interval (40 months) in our cohort. Conclusions: Molecular and immunophenotypic markers remain useful as adjuncts to clinical prognostication; however, as single parameters they are unable to dictate the timing of therapeutic intervention. The combined use of CD25 and IgVH mutational status may be clinically relevant to CLL prognostication while also providing insight into the biological pathways involved in disease progression.     

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.     

Comparison of flow cytometric methods for the measurement of ZAP‐70 expression in a routine diagnostic laboratory

Chronic lymphocytic leukaemia (CLL) follows a variable clinical course with patient survival ranging from only a few years despite treatment, to several decades in patients who may never require clinical intervention. Determination of the mutational status of a patient's immunoglobulin heavy chain variable region (Ig V_H) gene has been used to provide prognostic information, but this assay is not available in most laboratories. The discovery of the expression of the protein tyrosine kinase zeta‐associated protein (ZAP)‐70 in V_H‐unmutated CLL cases led to its proposal as a surrogate marker for V_H status. This study investigated the measurement of ZAP‐70 expression in CLL using different flow cytometric protocols. Two different antibodies and two different staining methods were compared. The Caltag ZAP‐70 antibody and Fix & Perm kit were the easiest to use and were the most sensitive and specific combination, with 91% concordance between ZAP‐70 expression and V_H status. Three patients (9%) were discordant (two V_H mutated/ZAP‐70 positive, and one V_H unmutated/ZAP‐70 negative). No correlation existed between CD38 and either ZAP‐70 expression or V_H status. Measurement of ZAP‐70 expression using the Caltag antibody/kit combination provides a standardized flow cytometric method that could be introduced into a routine CLL immunophenotyping panel in a clinical diagnostic laboratory.     

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.     

Quantification of ZAP-70 expression in chronic lymphocytic leukemia: T/B-cell ratio of mean fluorescence intensity provides stronger prognostic value than percentage of positive cells

Expression of ZAP-70 measured by flow cytometry belongs to the most powerful prognostic parameters in chronic lymphocytic leukemia (CLL). However, many technical factors such as setting of the positivity threshold may significantly influence results.. Quantification using mean fluorescent intensity (MFI) may eliminate the subjective error which is inevitable in the isotype control method. The aim of the present project was therefore to assess the prognostic significance of ZAP-70 using three different methods. Between 2005 and 2010 we measured ZAP-70 expression in 157 patients with CLL (108 males, 49 females, median age 60 years [range, 31-82]; low/intermediate/high Rai risk in 41/48/11%). Expression of ZAP-70 was determined by flow cytometry using phycoerythrin (PE)-conjugated monoclonal antibody, clone 1E7.2. Evaluation was performed by 1) percentage of positive cells compared to isotype control (cut-off 20%), 2) MFI ratio of T-cells/CLL cells (cut-off 3.0); 3) MFI ratio of ZAP-70/isotype control on CLL cells (cut-off 2.5). MFI method with T-cells/CLL cells ratio was the best in the identification of patients with unfavourable outcome: ZAP-70 positive patients had significantly shorter time to treatment (TTT, median 24 vs. 55 months, p=0.0001) and overall survival (OS, median 97 vs 174 months, p=0.0074). The differences in TTT a OS were not significant with the use of isotype percentage and MFI isotype methods. Combined analysis of ZAP-70 with CD38 expression or IgVH mutation status lead to identification of a subgroup with the longest TTT and OS (ZAP-70 and CD38 negative, p<0.0001 and p=0.012; ZAP-70 negative and mutated IgVH genes, p<0.0001 and p=0.0019). In conclusion, our results suggest that measurement of ZAP-70 expression in CLL by MFI using T-cells/CLL cells ratio might be the optimal method for accurate prediction of clinical course. Combined analysis of ZAP-70 with CD38 or IgVH mutation status further refined individual patient′s prognosis.     

Immunoglobulin gene segment usage, location and immunogenicity in mutated and unmutated chronic lymphocytic leukaemia

The mutational status of the variable region of the immunoglobulin heavy chain gene (IgV(H)) is an important prognostic marker in B-cell chronic lymphocytic leukaemia (B-CLL), with mutated patients having improved outcome. To examine the impact of mutational status on V(H), D(H), and J(H) gene segment location and immunogenicity, we analysed 375 IgH sequences from 356 patients with B-CLL. Although V(H) and D(H) gene usage was different in mutated compared to unmutated patients, there was no impact of gene location on frequency of use or clinical outcome. Surprisingly, somatic mutations did not increase the immunogenicity of the Ig, as assessed by predicted binding affinity of Ig-derived peptides to major histocompatibility Class I and Class II molecules. Even excluding patients using V(H)1-69, cases using the V(H)1 gene family had a poor outcome. Both mutated and unmutated CLL patients demonstrated evidence of antigen selection. The worst outcome was seen in the subset of 14 unmutated patients with similar HCDR3 amino acid sequence using V(H)1-69, D(H)3-3 and J(H)6, suggesting an antigen-driven process modulating the clinical course.     

FCRL2 expression predicts IGHV mutation status and clinical progression in chronic lymphocytic leukemia

CD38 and ZAP-70 are both useful prognostic markers for B-cell chronic lymphocytic leukemia (CLL), but are variably discordant with IGHV mutation status. A total of 5 human Fc receptor–like molecules (FCRL1-5) have tyrosine-based immunoregulatory potential and are expressed by B-lineage subpopulations. To determine their prognostic potential in CLL, FCRL expression was compared with IGHV mutation status, CD38 and ZAP-70 expression, and clinical features from 107 patients. FCRL1, FCRL2, FCRL3, and FCRL5 were found at markedly higher levels on CLL cells bearing mutated IGHV genes than on unmutated CLL cells or CD19⁺ polyclonal B lymphocytes. Univariate comparisons found that similar to CD38 and ZAP-70, FCRL expression was strongly associated with IGHV mutation status; however, only FCRL2 maintained independent predictive value by multivariate logistic analysis. Strikingly, FCRL2 demonstrated 94.4% concordance with IGHV mutation compared with 76.6% for CD38 and 80.4% for ZAP-70. Compared with other indicators, FCRL2 was also superior at predicting the time to first therapy; the median treatment-free interval was 15.5 years for patients with high FCRL2 expression compared with 3.75 years for FCRL2-low patients. Our studies indicate that FCRL2 has robust predictive value for determining IGHV gene mutation status and clinical progression and thus may further improve prognostic definition in CLL.     

Prognostic significance of combined analysis of ZAP-70 and CD38 in chronic lymphocytic leukemia

The clinical heterogeneity that characterizes chronic lymphocytic leukemia (CLL) poses critical questions concerning the identification of high risk patients. Unmutated IgV(H) genes, CD38 and ZAP-70 expression have emerged as the most useful tools in identifying aggressive CLL. The simultaneous expression of ZAP-70 and CD38 in 157 patients with CLL has been evaluated. Fifty-seven patients (36%) were positive for ZAP-70 and 46 patients (29%) were positive for CD38. Both molecules were highly correlated and predictive of the clinical course of the disease. According to the simultaneous evaluation of ZAP-70 and CD38, patients were divided into three groups. In 81 patients (52%), there was a negative concordance of both molecules (ZAP-70(-)/CD38(-)); in 27 patients (17%) there was a positive concordance (ZAP-70(+)/CD38(+)); in 49 patients (31%) there was a discordant expression (ZAP-70(+)/CD38(-) and ZAP-70(-)/CD38(+)). A comparison of the clinical and laboratory data showed in ZAP-70(+)/CD38(+) patients a significantly higher bone marrow and peripheral blood lymphocytosis, lower hemoglobin levels, more advanced clinical stage, and higher number of unmutated IgV(H) status with respect to the other two groups. Furthermore, ZAP-70(+)/CD38(+) patients displayed a much shorter treatment-free interval (median 12 months vs 42 months in discordant patients and not reached in ZAP-70(-)CD38(-) patients). These results prove that the concomitant evaluation of ZAP-70 and CD38 expression allows the separation of CLL patients in prognostic subgroups and suggest that their simultaneous assessment should become an integral component of the CLL diagnostic grid.     

ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) B cells that express unmutated immunoglobulin heavy-chain variable region genes (IgV(H)) generally express ZAP-70, in contrast to normal B cells or most CLL cases with mutated IgV(H). Following IgM ligation, ZAP-70+ CLL cells had significantly higher levels of phosphorylated p72(Syk), BLNK, and phospholipase-Cgamma (PLCgamma) and had greater[Ca2+]i flux than did ZAP-70-negative CLL cases, including unusual ZAP-70-negative cases with unmutated IgV(H). IgM ligation of ZAP-70-negative CLL B cells infected with an adenovirus vector encoding ZAP-70 induced significantly greater levels of phosphorylated p72(Syk), BLNK, and PLCgamma and had greater[Ca2+]i flux than did similarly stimulated, noninfected CLL cells or CLL cells infected with a control adenovirus vector. We conclude that expression of ZAP-70 in CLL allows for more effective IgM signaling in CLL B cells, a feature that could contribute to the relatively aggressive clinical behavior generally associated with CLL cells that express unmutated IgV(H).     

Relative value of ZAP-70, CD38, and immunoglobulin mutation status in predicting aggressive disease in chronic lymphocytic leukemia

Leukemia-cell expression of ZAP-70, CD38, or unmutated immunoglobulin heavy chain variable region genes (U-IGHV) each is associated with aggressive disease in patients with chronic lymphocytic leukemia (CLL). To assess the relative strength of each marker, we defined thresholds for designating a case as positive for CD38 or ZAP-70 in a test cohort of 307 patients and used these data-defined criteria to stratify patients in an independent cohort of 705 patients. Multivariable analysis revealed that ZAP-70 was the strongest risk factor. Knowledge of the IGHV mutation status or CD38 did not improve our ability to predict the time to first treatment except for ZAP-70-negative cases, which could be segregated into 2 groups of intermediate-risk or low-risk disease based on whether they expressed unmutated or mutated IGHV. ZAP-70 maintained its high relative prognostic value for the subset of patients with early-stage, asymptomatic disease, including patients evaluated within 1 year of diagnosis. Although it is premature to recommend therapy based on these risk factors, patients with ZAP-70-positive CLL cells should be monitored closely for disease progression as they have a median time from diagnosis to requiring initial therapy by standard criteria of approximately 3 years.     

Quantification of ZAP-70 mRNA by real-time PCR is a prognostic factor in chronic lymphocytic leukemia

Background

Chronic lymphocytic leukemia (CLL) is heterogeneous with respect to prognosis and clinical outcome. Mutational status of immunoglobulin heavy chain variable region (IGHV) appears to be a particularly strong prognostic marker, but it is difficult to perform in a routine clinical laboratory. ζ-chain-associated protein kinase 70?kDa (ZAP-70) protein detected by flow cytometry is a strong surrogate marker of IGHV mutational status; however, it suffers from the lack of standardization.

Methods

We investigated whether ZAP-70 mRNA expression level can be a prognostic factor in CLL. Real-time quantitative polymerase chain reaction was used to analyze ZAP-70 mRNA expression from 102 CLL patients.

Results

The expression of ZAP-70 mRNA was significantly associated with Binet stage (P?P?=?0.003), ZAP-70 protein (P?=?0.018), IGHV mutational status (P?=?0.038), and cytogenetic abnormality of del(17p13) or del(11q22.3) (P?=?0.037) in CLL patients. According to receiver operating characteristic curve analysis for ZAP-70 mRNA and ZAP-70 protein, positive ZAP-70 mRNA (P?=?0.006) was an adverse factor in determining the treatment free survival (TFS). In a multivariate Cox analysis of TFS, ZAP-70 mRNA is not ideal as an independent prognostic factor. However, ZAP-70 mRNA was statistically significant in predicting treatment response.

Conclusion

This study demonstrated the value of determination of ZAP-70 mRNA in providing useful prognostic information in CLL patients. However, ZAP-70 mRNA is not an independent prognostic factor.     

Significance of zeta-associated protein (ZAP-70) and CD38 expression in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a disease with a highly variable clinical course; some patients never need treatment, while others require intensive treatment early after diagnosis. Some new prognostic factors, such as immunoglobulin variable heavy chain (IgVH) mutational status, zeta-associated protein (ZAP-70) and CD38 expression in leukemic cells were introduced to identify attenuated versus progressive types of CLL bearing the potential to facilitate risk-adapted treatment strategies. So, the aim of this work is to evaluate the clinical value of ZAP-70 and CD38 as predictors of disease progression. We assessed the expression of these markers by flowcytometry in 38 patients with CLL and correlated their levels with genetic abnormalities detected by fluorescence in situ hybridization (FISH) and the clinical outcome. We found that 18 patients (47.4 %) were positive for ZAP-70 (> or = 20%) and 16 patients (42.1%) were positive for CD38 (> or = 20%). Positive ZAP-70 and CD38 status were associated with an unfavorable clinical course including high leukocytic count, lymphocytosis, high lactate dehydrogenase (LDH) serum level, advanced disease stage, trisomy 12 and del (11q); negative ZAP-70 and CD38 status were correlated with del (13q). The treatment-free survival time was 30 months for ZAP-70-positive patients and 18 months for ZAP-70-nagative patients (p < 0.01). Combined analysis of ZAP-70 and CD38 yielded discordant results in 10 patients (26.3 %), whereas 16 patients (42.1%) were concordantly negative and 12 patients (31.6%) were concordantly positive for ZAP-70 and CD38 expression. Median treatment-free survival times in patients whose leukemic cells were ZAP-70+CD38+ was 27 months as compared to 100 months in patients with a ZAP-70(-)CD38(-) status. In patients with discordant ZAP-70/CD38 results, the median treatment-free survival time was 40 months. Thus, ZAP-70 and CD38 expression analyses provide complementary prognostic information and allow distinguishing the patients groups with the most favorable prognosis as well as those with the worst. The current findings suggest that both ZAP-70 and CD38 protein expression should be assessed in patients with CLL for the definition of prognostic subgroups.     

Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia

CD49d/alpha4-integrin is variably expressed in chronic lymphocytic leukemia (CLL). We evaluated its relevance as independent prognosticator for overall survival and time to treatment (TTT) in a series of 303 (232 for TTT) CLLs, in comparison with other biologic or clinical prognosticators (CD38, ZAP-70, immunoglobulin variable heavy chain (IGHV) gene status, cytogenetic abnormalities, soluble CD23, beta2-microglobulin, Rai staging). Flow cytometric detection of CD49d was stable and reproducible, and the chosen cut-off (30% CLL cells) easily discriminated CD49dlow from CD49dhigh cases. CD49d, whose expression was strongly associated with that of CD38 (P<.001) and ZAP-70 (P<.001), or with IGHV mutations (P<.001), was independent prognosticator for overall survival along with IGHV mutational status (CD49d hazard ratio, HRCD49d=3.52, P=.02; HRIGHV=6.53, P<.001) or, if this parameter was omitted, with ZAP-70 (HRCD49d=3.72, P=.002; HRZAP-70=3.32, P=.009). CD49d was also a prognosticator for TTT (HR=1.74, P=.007) and refined the impact of all the other factors. Notably, a CD49dhigh phenotype, although not changing the outcome of good prognosis (ZAP-70low, mutated IGHV) CLL, was necessary to correctly prognosticate the shorter TTT of ZAP-70high (HR=3.12; P=.023) or unmutated IGHV (HR=2.95; P=.002) cases. These findings support the introduction of CD49d detection in routine prognostic assessment of CLL patients, and suggest both pathogenetic and therapeutic implications for CD49d expression in CLL.