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.     

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.     

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.     

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.     

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.     

New prognostic markers in chronic lymphocytic leukemia

The prognosis of patients with chronic lymphocytic leukemia (CLL) is extremely variable. Prognostication of patients with CLL has been classically based on clinical parameters. In the last few years, several biologic markers such as cytogenetics, IgVH mutations, CD38 and ZAP-70 expression in leukemic cells have shown to offer important prognostic information. However, before being incorporated into daily practice these markers require standardization and validation in prospective trials. Meanwhile, prognosis of patients with CLL should remain to be based on clinical stages and other easily obtainable clinical parameters. An important area of research is the identification of markers useful for predicting response to therapy. Among them, 17p- reflecting p53 abnormalities is particularly important. Also relevant is 11q- pointing out to ATM defects. The correlation of IgVH mutations, ZAP-70 and CD38 expression with response is unclear and needs further investigation. Finally, there is increasing evidence that response to therapy, particularly when all measurable disease is eradicated, is associated with longer survival.     

CD38 as a prognostic marker in CLL

Cell-surface expression of CD38 in CLL has been recognised recently as a marker of progressive disease and poor outcome. In contrast to traditional staging systems, CD38 is able to identify progressive cases at an early stage. Measurement of CD38, in conjunction with other novel prognostic factors such as p53 and ZAP-70 helps to identify patients who might benefit from early and more intensive therapy. In addition, CD38 positivity can predict unmutated IgVH gene mutation status in most cases. These features, together with its easy applicability, render CD38 a valuable tool in the routine diagnostics of CLL. Questions remaining to be clarified about CD38 include the incidence and significance of its variations during the course of the disease, the optimal method to define CD38 positivity and the impact of different methodologies on results. Only after these issues are resolved can the definitive place of CD38 be defined in the diagnostics of CLL.     

CD38 as a prognostic marker in CLL

Cell-surface expression of CD38 in CLL has been recognised recently as a marker of progressive disease and poor outcome. In contrast to traditional staging systems, CD38 is able to identify progressive cases at an early stage. Measurement of CD38, in conjunction with other novel prognostic factors such as p53 and ZAP-70 helps to identify patients who might benefit from early and more intensive therapy. In addition, CD38 positivity can predict unmutated IgVH gene mutation status in most cases. These features, together with its easy applicability, render CD38 a valuable tool in the routine diagnostics of CLL. Questions remaining to be clarified about CD38 include the incidence and significance of its variations during the course of the disease, the optimal method to define CD38 positivity and the impact of different methodologies on results. Only after these issues are resolved can the definitive place of CD38 be defined in the diagnostics of CLL.     

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.     

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.     

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

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

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.     

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).     

Mcl-1 expression has in vitro and in vivo significance in chronic lymphocytic leukemia and is associated with other poor prognostic markers

Bcl-2 family proteins play a critical role in the regulation of apoptosis in chronic lymphocytic leukemia (CLL). However, their association with established prognostic markers is unknown. In this study, we analyzed the expression of Bcl-2, Bax, and Mcl-1 in 185 CLL patients and evaluated their relationship with other prognostic markers, in vitro sensitivity to fludarabine, and clinical outcome. Mcl-1 expression was significantly correlated with stage of disease (P < .001), lymphocyte doubling time (P = .01), V(H) gene mutation status (P < .001), CD38 expression (P < .001), and ZAP-70 expression (P = .003). In addition, Mcl-1 and Mcl-1/Bax ratios showed strong correlations with in vitro resistance to fludarabine (P = .005 and P < .001, respectively). Furthermore, elevated Mcl-1 expression and Mcl-1/Bax ratios were predictive of time to first treatment in the whole cohort (P < .001 and P < .001, respectively) and in stage A patients only (P = .002 and P = .001, respectively). Taken together, our data show that Mcl-1 is a key controller of in vitro drug resistance and is an important regulator of disease progression and outcome in CLL. It therefore represents a promising therapeutic target in this incurable condition. The close correlation between Mcl-1 expression and V(H) gene mutation status, CD38 expression, and ZAP-70 expression offers a biologic explanation for their association with adverse prognosis.     

Insight into the pathogenesis of chronic lymphocytic leukemia (CLL) through analysis of IgVH gene usage and mutation status in familial CLL

To address the proposition that familial B-cell chronic lymphocytic leukemia (CLL) may exhibit a more restricted phenotype than sporadic CLL with respect to immunoglobulin gene usage or ontogenic development, we compared immunoglobulin (Ig) heavy chain variable region (VH) gene usage and IgVH mutation status in 327 patients with CLL from 214 families with 724 patients with sporadic cases. The frequency of mutated CLL was higher in familial CLL (P < .001), and there was evidence of intrafamilial concordance in mutation status (P < .001). The repertoire and frequency of IgVH usage was, however, not significantly different between familial and sporadic CLL. Furthermore, IgVH usage was not correlated between affected members of the same family. These observations provide evidence that familial CLL is essentially indistinguishable from sporadic CLL, favoring a genetic basis to disease development in general rather than a simple environmental etiology.     

Graft-versus-leukaemia effect after non-myeloablative haematopoietic transplantation can overcome the unfavourable expression of ZAP-70 in refractory chronic lymphocytic leukaemia

ZAP-70 (zeta-chain-associated protein 70 kDa) expression is associated with poor prognosis in patients with chronic lymphocytic leukaemia (CLL). This study evaluated the efficacy of non-myeloablative allogeneic stem cell transplantation in patients with advanced CLL and assessed the impact of ZAP-70 expression on the outcome. Thirty-nine sequential patients were included. All had previously been treated with fludarabine. All patients received a preparative regimen of fludarabine (30 mg/m(2)/d for 3 d), intravenous cyclophosphamide (750 mg/m(2)/d for 3 d), and high-dose rituximab. Immunohistochemical techniques on marrow biopsy samples were used to determine that ZAP-70 was expressed in 25 patients, whereas 13 other patients were ZAP-70 negative, and one was of indeterminate status. With a median follow-up time of 27 months, the estimated overall survival and current progression-free survival (CPFS) rates at 4 years were 48% and 44% respectively. Patients who were ZAP-70 positive had 56% survival, and their CPFS rate increased from 30% to 53% after a donor lymphocyte infusion. Multivariate analysis indicated that chemorefractory disease and mixed T cell chimerism at day 90, but not ZAP-70 positivity, were associated with the risk of disease progression after transplantation. These results demonstrate a potent graft-versus-leukaemia effect that can overcome the adverse prognostic effect of ZAP-70 expression.