ZAP-70 Expression In Chronic Lymphocytic Leukemia Research Articles

Expression of ZAP 70 in Chronic Lymphocytic Leukemia (CLL) and its Correlation with Clinical Stages

Objective: To ascertain the levels of ZAP-70 expression within the context of Chronic Lymphocytic Leukemia (CLL) patients and to subsequently analyze the potential correlations between ZAP-70 expression and the clinical staging of the disease. Study Design & Setting: Cross-sectional, descriptive study. Department of Hematology, Armed Forces Institute of Pathology (AFIP), Rawalpindi from July 2021 to December 2021. Methodology: We included 73 CLL patients of various ages and both genders. and patients having lymphoproliferative disorders other than CLL were excluded. Sysmex XN-3000 was used to do complete blood counts. Diagnosis of CLL was confirmed immunophenotypically by flow cytometry. Samples were processed by standard methods for ZAP70 analysis. Descriptive statistics were expressed in terms of mean ± standard deviation (SD). A Chi-square test was conducted, with a significance level of p-value =0.05 being considered significant. Results: Among 73 patients, 24 were females (32.9%) and 49 were males (67.1%). Females had a mean age of 69.00±9.47 years, whereas males had a mean age of 65.73±11.12 years. ZAP-70 expression was positive in 8 (11%) and negative in 65 (89.0%) cases. The expression of ZAP-70 in CLL and its correlation with Binet Stages was significant (p=0.002). ZAP- 70 expression in males and females showed no significant difference (p 0.059). ZAP-70 analysis showed no significant difference in patients with age more than 60 years from = 60 years of age (p= 0.275) Conclusion: ZAP-70 gene expression should be used in routine laboratory settings and can be correlated with the clinical stages of CLL

Heat Shock Protein 90 (Hsp90) Activity Status Is An Independent Prognostic Marker in Chronic Lymphocytic Leukemia (CLL) and Correlates with IgVH Mutational Status, ZAP-70 Expression and Time to First Treatment

Abstract 2544The clinical presentation of chronic lymphocytic leukemia (CLL) is variable and includes at least two distinct clinical subtypes: indolent or progressive disease. Several prognostic markers such as the mutational status of the IgVH genes or ZAP-70 expression identify patients that will have more aggressive clinical courses. We have previously reported that ZAP-70 expression in CLL requires the support and stability provided by the Hsp90 activated complex (Hsp90-AC) and that Hsp90 inhibitors induce apoptosis in CLL cells preferentially in patients with high–risk/rapid progressive disease.Hsp90 requires the conformation of a multimeric complex with other co-chaperones (Hop, p23) to become active and function as a chaperone for mutated, over-expressed, misfolded or constitutively activated proteins. However, to measure Hsp90-AC activity is challenging and requires protein assays that are semi-quantitative and not always can provide a functional read out. To address this problem we designed a test to measure Hsp90-AC by taking advantage of the higher avidity of this complex to bind Hsp90 inhibitors such as 17-AAG. We hypothesize that Hsp90-AC activity is an independent prognostic marker of disease progression in CLL and to test this we measured the ability of 17-AAG to inhibit Hsp90-AC and promote apoptosis in vitro in a cohort of previously untreated CLL patients. This evaluation was performed in samples collected at the time of diagnosis and the results were correlated with the time from diagnosis to first treatment (t-Tx1) as well as the presence of other known prognostic markers.Ninety-five previously untreated CLL patients were included in this cohort. In vitro sensitivity/apoptosis to Hsp90-AC inhibition using 17-AAG (1μg/ml) was measured by flow cytometry using DiOC6 and PI after 48 hours of incubation. Only samples with more than 60% viability were included and we selected a cut-point of ≥ 53% induction of apoptosis to determine if a sample was sensitive or not to Hsp90-AC inhibition (This cut-point was selected using a ROC analysis for optimal diagnostic performance).We found that 37 patients (39%) were sensitive to 17-AAG. These patient had a median time from diagnosis to first treatment (t-Tx1) of 7 years compared to 4.2 years in patients that were resistant to 17-AAG (p=0.04). In addition, 37 patients (39%) had unmutated IgVH genes with a median t-Tx1 of 8.7 years compared to 4.2 years in patients with mutated IgVH genes (p=0.02) and 41 patients (43%) had high levels of ZAP-70 expression (>20% by flow cytometry) and this group of patients had a median t-Tx1 of 7 years vs. 4.2 years in ZAP-70 negative patients (p=0.03). We evaluated the association between the degree of Hsp90-AC inhibition (17-AGG sensitivity) and IgVH mutational status and found a strong correlation between these two variables with shorter t-Tx1 in patients that were IgVH unmutated and sensitive to Hsp90 inhibitor (p=0.0003). We found a similar strong correlation between ZAP-70 expression and sensitivity to Hsp90-AC inhibitor (p=0.003). In our patient cohort, we saw that IgVH, ZAP-70 and Hsp90 inhibitor sensitivity are independent prognostic markers in CLL (Cox regression, p=0.002). We found that the sensibility and specificity of ZAP-70 test for high-risk CLL (sensitivity 81.1% [95% IC 67–95%] and specificity 77.6%[95% IC 65–90%]) was increased when the Hsp90-AC inhibitor sensitivity test was added (sensitivity 87% [95% IC 71–100%] and specificity 91%[95% IC 79–100%]).In conclusion, in vitro inhibition of Hsp90-AC in CLL using 17-AAG is an independent prognostic maker and strong predictor of the need for treatment. Hsp90-AC inhibition showed a high correlation with IgVH mutational status and ZAP-70 expression. Additionally, patients sensitive to 17-AAG whose leukemic cells also highly express ZAP-70 appear to have the worst prognosis with the shortest t-Tx1. Our data suggest that in vitro sensitivity to 17-AAG at the time of diagnosis serves as a quantitative surrogate marker for Hsp90-AC activity and correlates with disease progression in CLL. This is the first time that such correlation has been established in a cohort of cancer patients and shows the relevance of the Hsp90 chaperone system in cancer biology and disease progression. Disclosures:No relevant conflicts of interest to declare.

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.

Influence of ZAP-70 Expression On Migration of Chronic Lymphocytic Leukemia (CLL) and Lymphoma Cells.

Abstract 2345Poster Board II-322ZAP-70 (ξ-associated protein) is a tyrosine kinase (PTK) of the Syk/ZAP family normally expressed in T and NK cells. Increased ZAP-70 expression in CLL correlates with unmutated IgVH genes, a short time of progression, and a short survival. Mechanisms by which increased ZAP-70 protein expression can influence clinical outcome are not fully understood. After B-cell receptor (BCR) stimulation, activated ZAP-70 can cooperate with Syk, the tyrosine kinase of the BCR signal pathway normally expressed in B-cells, thus inducing activation of the CLL cells. Stimulation of the BCR leads to the triggering of Syk protein kinase to the cytoplasmatic tails of the receptor initiating subsequent activation of critical effector enzymes such as PI3K and PLCγ2. The PI3K/Akt pathway is related to survival and protection from BCR-induced apoptosi. Against this background, we analyzed the functional consequences of abnormal expression of ZAP-70 protein in B-lymphoma cell lines that normally does not express ZAP-70, particularly analyzing the impact of increased ZAP-70 expression oncell metabolism and cell migration. Mec-1 (CLL), Raji and Ramos (Burkitt) cell lines were stably transfected with the ZAP-70 expression vector pEGFP-ZAP-70 as well as the control pEGFP. BCR stimulation was induced in the stable Ramos cell line by IgM at several time points, and phosphorylation of downstream proteins was analyzed by Western Blot. Proliferation, cell cycle, calcium flux, adhesion molecules and cell migration were analyzed in all stable cell lines after ZAP-70 phosphorylation. After IgM stimulation, in Ramos cell line phosphorylation of ZAP-70 was observed at residue Y319 and, simultaneously, phospho-Syk expression was reduced. In addition, Erk protein was strongly activated in ZAP-70 transfected cell line compared to the control cell line, this activation lasting more than 24 hours. Notably, calcium flux detected by flow cytometry was higher in ZAP-70 transfected Ramos. Migration experiments showed that after IgM stimulation, transfected ZAP-70 Ramos migrated significantly more rapidly than cells transfected with the vector alone. Of note, after IgM stimulation, ZAP-70 transfected Ramos cells expressed a higher number of adhesion molecules (CXCR7 and others) on surface membrane than non-transfected cells. This effect on migration stimulation was also observed with the Raji cell line transfected with ZAP-70. In conclusion, addition of ZAP-70 expression to a heterologous B-cell system enhances BCR downstream signalling, calcium mobilization, and migration. Ongoing studies are determining the influence on the gene expression profile dependent of ZAP-70 expression. Altogheter, these data suggest that additional ZAP-70 expression in CLL and other lymphoproliferative disorders enhances tumoral activity, this explaining in part the poor clinical prognosis of patients with increased expression of ZAP-70. Disclosures:No relevant conflicts of interest to declare.

ZAP-70 Intron-1 DNA Methylation Status: A Simplified Method of Determination by Pyrosequencing in B Chronic Lymphocytic Leukemia.

ZAP-70 expression is a strong prognostic marker in chronic lymphocytic leukemia (CLL). However, standardization of ZAP-70 using flow cytometry is problematic. In previous study, ZAP-70 methylation status, assessed by COBRA methodology, was found to be nicely correlated with ZAP-70 expression in CLL (Corcoran et al. Haematologica 2005; 90:1078–1088). However, this technique is poorly suitable for routine use. We developed a new quantitative method for ZAP-70 methylation that simplifies this approach. Pyrosequencing is a sequencing-by-synthesis method that uses the release of pyrophosphate (Ppi) molecule during incorporation of nucleotides. Released Ppi are quantitatively converted into a light flash by enzymatic cascade. These light signals are measured by a charge-coupled device camera and are displayed as peaks in a pyrogram. Pyrosequencing combines the ability of direct quantitative sequencing, reproducibility, speed, ease-of-use. It has many advantages compared to other techniques for studies of methylation as bisulfite sequencing, COBRA, methylspecific PCR (MSP) and quantitative-MSP (Q-MSP). Samples were obtained from blood for 115 CLL patients and from paraffin embedded tissue after lymph node biopsy for 8 patients with 2 CLL, 2 MCL and 4 DLBCL. All patients were untreated at the time of sampling. Binet stage at diagnosis for the 117 CLL were stage A (114 pts), stage B (2pts), stage C (1pt). DNA from peripheral blood cells and from paraffin-tissue samples was treated by bisulfite and the ZAP-70 intron1-exon2 region was amplified using ZAPDN1-F and ZAPDN1-R primers. The percentage methylation was calculated as the average CpG methylation per clone and of all the clones sequenced for each sample. We found that methylation in four CpG pairs (C-223, C-243, C-254, C267) in the first intron of ZAP-70 highly correlated with repression of ZAP-70, as determined by western blot and real time PCR. Calibration mixture of DNAs with known methylation were analyzed. As expected, results showed that T-lymphocytes DNA is umethylated with a mean of 4% [range 2–12%, mean (SD) 0.41%] whereas M.SssI treated DNA was nearly fully methylated in the 4 CpG sites with a mean of 89% [range: 73 to 100%, mean (SD): 1,54%]. For each patient, biotinylated single-strand DNA fragments were generated, amplified by PCR and sequenced using an automated pyrosequencing instrument, PSQTM 96 MA (Biotage). A 50% methylation level cut-off easily discriminates ZAP-70 positive from negative CLL cells. There was an excellent correlation between methylation quantification results observed with bisulfite sequencing and pyrosequencing for the 4 CpG sites (r2>0,95). DNA extracted from tissue paraffin embedded tissue (2 CLL, 2 MCL, 4 DLBCL) show the same quality of pyrograms. Sensibility of pyrosequencing is about 5%. Additionally, we found an excellent correlation of ZAP-70 hypermethylation status with IgVH mutational status (p<0.0001) and CD38 expression (p<0.0001). Patients with unmethylated ZAP-70 had shorter median overall survival (80 months/not reached, p=0.0014) and time to treatment (24 months/not reached, p<0.0001). The pyrosequencing assay has several advantages: it is straight-forward to perform, provides high reproducibility, is independent of T-lymphocytes contamination, needs small quantity of DNA, is usable from tissue paraffin or frozen cells and do not express the uncertainties associated with the use of ZAP-70 protein expression. In addition, pyrosequencing delivers results faster and at lower cost than IgVH sequencing and is more suitable for routine use.

Zap-70 positive chronic lymphocytic leukemia co-existing with Jak 2 V671F positive essential thrombocythemia: A common defective stem cell?

Essential thrombocythemia (ET) co-existing with chronic lymphocytic leukemia (CLL) is extremely rare. We report two cases of ET with Jak 2 V617F in Zap-70+ CLL. ET is a myeloproliferative stem cell disease. Zap-70 expression in CLL correlates with non-mutated immunoglobulin genes. The occurrence of a less mature CLL in patients with a pluripotential stem cell disease raises the possibility that an initial “trigger hit” occurred in a pre-Jak 2 common early progenitor in these patients. Subsequent additional molecular events accumulated independently following myeloid and lymphoid differentiation, leading to the development of two diseases of likely identical origin but different lineages.

ZAP-70 Expression in Chronic Lymphocytic Leukemia Is Associated with Altered Signal Transduction.

B-cell chronic lymphocytic leukemia (CLL) is a malignancy of mature B-cells with a variable clinical course. Given the heterogeneity of disease course, identification of prognostic factors is imperative. One recently identified negative prognostic indicator in CLL is the T-cell tyrosine kinase ZAP70. ZAP70 is analogous to, and can functionally substitute for, the B-cell tyrosine kinase Syk and is activated following B cell receptor (BCR) crosslinking in CLL cells. However, the role of ZAP70 expression in CLL remains elusive. Utilizing flow cytometric analyses of signal transduction molecules, this study examined if ZAP70 expression affects signal transduction in CLL. Peripheral blood samples from ZAP70+ and ZAP70− CLL patients were analyzed. The levels of phosphorylated ERK, STAT5, and S6 (p-ERK, p-STAT5, p-S6) were measured by flow cytometry with and without BCR stimulation. Kinase inhibitors were used to verify the measured phosphorlyated protein levels. Increased constitutive levels of p-ERK, p-STAT5, and p-S6 were seen in ZAP70+ CLL cells as compared to ZAP70− CLL cells. BCR crosslinking gave variable responses in both ZAP70+ and ZAP70− CLL. The majority of ZAP70− cases showed no significant activation or a predominant activation of ERK with rare p-S6 activation. ZAP70+ cells showed activation of either, or both, ERK and S6 pathways and fewer showing no activation. In summary, though the role of ZAP70 in B cell signaling is poorly understood, expression of ZAP70 in CLL correlates with a constitutively active phenotype. Additionally, differential response in ZAP70+ versus ZAP70− cases following BCR crosslinking suggests a possible modulatory role of the BCR pathway. Current studies are underway to determine if these measures carry prognostic information and if the varying signal responses to BCR stimulation correlate with reported differences in apoptotic versus proliferative response.

Correlation of Flow Cytometrically Determined Expression of ZAP-70 Using Two Different Antibodies with IgVH Mutation Status and Cytogenetics in 539 Patients with Chronic Lymphocytic Leukemia.

The clinical course of chronic lymphocytic leukemia (CLL) is largely heterogeneous. It may be estimated based on clinical and laboratory findings the most important ones of which include chromosomal aberrations, the IgVH mutation status, and the expression of ZAP-70. The optimum procedure of ZAP-70 assessment including selection of antibody clone and the method of analysis still needs to be established. The validation of ZAP-70 has been performed using the course of the disease as well as using the IgVH mutation status. To further clarify the role of ZAP-70 expression in CLL we prospectively analyzed ZAP-70 expression, IgVH mutation status, and chromosome aberrations in peripheral blood and bone marrow samples of 539 patients with CLL. For the flow cytometric assessment of ZAP-70 expression the antibody clones 1E7.2 (Caltag, n=523) and SBZAP (Immunotech, n=81) have been used. IgVH mutation status has been analyzed in 407 cases and chromosome aberrations in 416 cases applying FISH with probes for del(6q), del(11q), +12, del(13q), del(17p), and t(11;14). ZAP-70 expression has been calculated as percentage of positive cells using normal T-lymphocytes as positive controls as well as using the ratio of mean fluorescence intensity (MFI) for ZAP-70 between T-lymphocytes and leukemic B-lymphocytes. Significant correlations were found between the percentages of ZAP-70 positive cells and IgVH homology (1E7.2, p<0.001, r=0.239; SBZAP, p=0.003, r=0.426). Accordingly, patients with mutated and unmutated IgVH status (98% homology used as cut-off) significantly differed in the percentages of ZAP-70 positive cells (1E7.2, mean 32% vs. 46%, p<0.001; SBZAP, mean 7% vs. 29%, p<0.001). Applying a cut-off level of 20% for positivity of ZAP-70 expression significant correlations (ZAP-70 positive and IgVH unmutated and vice versa) were found for 1E7.2 (p<0.001, rate of concordant cases 59.0%) and SBZAP (p=0.002, 80.0%). Even stronger correlations have been found, however, comparing the MFI ratios for ZAP-70 with the IgVH mutation status: 1E7.2, p<0.001, r=−0.283; SBZAP, p<0.001, r=−0.494. The respective differences between cases with mutated and unmutated IgVH status were: 1E7.2, mean MFI ratio 3.45 vs. 2.67, p=0.001; SBZAP, mean MFI ratio 6.18 vs. 2.86, p<0.001. The ratio of MFI between leukemic and normal B-lymphocytes did not correlate with the IgVH mutation status. No associations of ZAP-70 expression with chromosomal aberrations have been found. The results of the present study1) confirm the strong correlation between ZAP-70 expression and IgVH mutation status,2) confirm the independence of ZAP-70 expression from chromosome aberrations,3) argue in favor of analyzing ZAP-70 expression as MFI ratio between T-lymphocytes and leukemic B-lymphocytes, and4) suggest that the SBZAP clone has a significant potential in the diagnostic work-up of CLL.Further studies are warranted to validate the role of the MFI ratio approach and of the SBZAP antibody in the clinical setting.

Expression of ZAP-70 in Chronic Lymphocytic Leukemia Cells Enhances CD79b Phosphorylation Following Surface IgM Ligation.

CD79b is B-cell surface molecule that non-covalently associates with CD79a and surface immunoglobulin (sIg), which together serve as the B-cell receptor complex (BCR). Both CD79a and CD79b have cytosolic immunoreceptor tyrosine-based activation motifs (ITAMs) that can become phosphorylated following sIg ligation, thereby allowing for recruitment to the BCR complex of cytosolic kinases, such as p72Syk , which then can initiate downstream intracellular signaling events. Compared to normal B cells, chronic lymphocytic leukemia (CLL) B cells typically expresses low levels of CD79b, which is speculated to contribute to the relatively poor capacity of CLL cells to initiate intracellular signaling following BCR ligation despite having apparently adequate levels of p72Syk. BCR signaling in CLL cells can be enhanced by expression of the zeta-associated protein of 70 kD (ZAP-70), a tyrosine kinase that initially was identified in T cells, where it plays a critical role in the phosphorylation of ITAMs of the accessory molecules of the T-cell receptor (TCR) complex for antigen following TCR ligation. We investigated for phosphorylation of CD79b following BCR ligation with F(ab)2 anti- μ antibody in CLL cell samples that did or did not express ZAP-70. All CLL cell samples expressed similar amounts of surface IgM and p72Syk, as assessed via flow cytometry and immunoblot analysis. Within 10 minutes after treatment with anti-μ the CLL cell samples that expressed ZAP-70 (n = 28) experienced a mean increase in phosphorylation of CD79b of 21.5% (± 14.0% S.D.), which was significantly greater than the 7.5% increase (± 7.9% S.D.) experienced by similarly treated CLL cell samples that did not express ZAP-70 (n = 19) (P< 0.01). Immune precipitation studies demonstrated association of CD79b with p72Syk in CLL B cells. CLL cell samples (n = 5) lacking expression of ZAP-70 were transfected with a control vector or an expression vector encoding ZAP-70, allowing us to examine the effect that engineered-expression of ZAP-70 has on CD79 phosphorylation following treatment with anti-μ. Anti-μ treatment induced significantly higher mean levels of CD79b phosphorylation in CLL samples made to express ZAP-70 (33% ± 16%) than in control mock-transfected CLL cells (4% ± 2%). This also was associated with enhanced anti-μ induced phosphorylation of p72Syk. We conclude that expression of ZAP-70 in CLL B cells enhances phosphorylation of the accessory molecules in the BCR complex following sIg ligation, potentially allowing for improved recruitment of cytosolic kinases and adapter proteins to these accessory molecules for enhanced BCR signaling.

Clinical utility of assessing ZAP‐70 and CD38 in chronic lymphocytic leukemia

Clinical utility of assessing ZAP‐70 and CD38 in chronic lymphocytic leukemia

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.

B-Cell Scaffold Protein with Ankyrin Repeats (BANK1) Is Differentially Expressed in ZAP-70 Negative Compared to ZAP-70 Positive Chronic Lymphocytic Leukemia Cells.

The lack of somatic mutations of the immunoglobulin variable heavy chain (IgVH) gene has been established as poor prognostic marker for chronic lymphocytic leukemia (CLL) patients at early stage disease. Expression of the non receptor tyrosine kinase zeta chain associated protein (ZAP-70) was proposed as a surrogate marker for an unmutated IgVH, however, up to 30% discordant samples have been reported depending on the respective study. B cell receptor (BCR) mediated signaling is enhanced by ZAP-70 expression in CLL cells in vitro and ZAP-70 expression also tends to decrease the time from diagnosis to treatment irrespective of the IgVH status. Therefore, we wanted to identify differentially expressed genes between the ZAP-70 positive and negative CLLs by gene expression profiling of peripheral blood mononuclear cells (PBMCs) using Affymetrix microarrays (HG-U133 Plus 2.0). ZAP-70 expression was analyzed by quantitative real time PCR of CD19 purified (purity > 99%) PBMCs (n=62) using a LightCycler instrument. Expression of ZAP-70 mRNA was normalized against the housekeeping gene ABL and a relative quantitation against Jurkat T cells as a calibrator was performed. Results are expressed as normalized ratio and a cut-off of 0.5 normalized ratio gave the best correlation to the IgVH status with 77% concordant samples between ZAP-70 expression and the IgVH status. The discordant samples consisted of 5 unmutated IgVHs in the ZAP-70 negative group and 9 mutated in the ZAP-70 positive group. In a second step PBMCs of the same samples were analyzed by gene expression profiling and differentially expressed genes were identified by t-test. Among the two best genes that could be used in a classification algorithm (SVM) to distinguish between the 2 subsets with 92% accuracy were ZAP-70 and B cell scaffold protein with ankyrin repeats (BANK1). The expression of BANK1 was increased 3–4-fold in the ZAP-70 negative compared to the ZAP-70 positive CLL subset (P = 0,001). In the literature, BANK1 has been identified in human BCR expressing B cells and seems to be B cell restricted. In B cells the scaffolding protein BANK1 enhances BCR-mediated Ca2+-signaling, a signaling pathway that is also enhanced by ZAP-70 expression in CLL B cells. Based on these data we show that increased BANK1 expression correlates with a ZAP-70 negative status in CLL B cells. The functional consequences of BANK1 expression in the ZAP-70 negative subset of CLL B cells, which are usually associated with a more favorable prognosis, still need to be established further.

Surface Antigens Identified by Antibody Microarray That Correlate with IgVH Mutational Status and ZAP70 Expression in CLL.

A new method has been developed to evaluate leukemias based on binding of cells in suspension to a microarray of cluster of differentiation (CD) monoclonal antibodies (mAbs) immobilized on nitrocellulose film on a microscope slide. Cell binding is proportional to antigen expression. This method allows simultaneous evaluation of 88 different CD antigens and is feasible with homogeneous populations of mononuclear cells obtained from blood of patients with leukemia. This methodology is potentially useful in diagnosis and classification of different leukemias and lymphomas and it may further aid in identifying and distinguishing subgroups of patients within a particular diagnosis. We utilized this methodology to assess its effectiveness in distinguishing prognostic subgroups in patients with chronic lymphocytic leukemia (CLL), those with mutated versus unmutated IgVH genes and those with <20% ZAP70+ versus >20% ZAP70+ cells. Unmutated IgVH genes were defined as >98% homologous to germline. To do this we identified 101 unselected samples from CLL patients for which IgVH mutational status and/or ZAP70 expression were known. Both IgVH status and ZAP70 expression were known for 70, in 6 only IgVH status, and in 25 only ZAP70 was known. IgVH mutational status and ZAP70 expression were concordant in 62/70 cases. Patient characteristics (median and range) were as follows: age=59yrs(37–82); abs lym=23k/μL(3.6–123); β2M=2.3mg/L(1.2–9.5); # prior treatments= 0(0–6). 20 had Rai low-risk; 67 had Rai-intermediate-risk, and 14 had Rai high-risk disease; 78 were previously untreated. Reduced space linear discrimination analysis and empirical Bayes moderated t-test was used to evaluate relationships between microarray binding and patients with “good prognosis” (IgVH mutated or ZAP70-) and “poor prognosis” (IgVH unmutated or ZAP70+). Empirical Bayes analysis was used to identify CD antigens with significant differential expression between the “good prognosis” and “poor prognosis” groups. Although there was not a significant difference in overall expression between the two groups, there were antibodies that had significantly different levels of binding. For the IgVH unmutated group, increased binding was noted with CD95, CD38, CD2, CD13, CD11a, CD86, CD25, CD9, CD22 (p<.05; FDR-adjusted). For the ZAP70+ group, increased binding was noted for CD38, CD2, CD95, CD49d, CD79b, kappa, and CD11a (pFDR<.05). In the IgVH/ZAP70 concordant cases, the following had increased binding with the “poor prognosis” group (IgVH unmutated/ZAP70+): CD38, CD95, CD2, CD13, CD24, CD9, and CD11a (pFDR<.05). There is clear evidence of differential binding on the mAb microarray between prognostic groups, although it is not yet possible to accurately predict ZAP70 or IgVH mutation status based on microarray results. Work continues to correlate these findings with flow cytometry and with other meaningful prognostic factors such as β2M; Rai stage, etc. In addition, follow-up continues to assess correlations between microarray results and significant clinical endpoints including survival. This is a simple immunomicroarray method that may be useful and important to enhance the classification and prognostic assessment of patients with lymphoid malignancies, including CLL, based on correlations between an extensive immunophenotype and clinical endpoints.

The Expression of Human Leukocyte Formin Protein, a New Protein That Associates with AKT, Is Correlated with the Expression of ZAP-70 in CLL.

Recently, we have cloned a new human gene (GenBank Accession No. AY278319), belonging to the formin family. This new gene, called for us human leukocyte formin, presents the common domains found in formin-related proteins: FH1, FH2 and FH3 domains. Western Blotting analysis has demonstrated that the protein encoded by this gene is overexpressed in lymphoid malignancies and cancer cell lines. Based on this pattern of expression, our objective was to investigate the expression of human leukocyte formin protein, by Western blotting analysis, in mononuclear cells from chronic lymphocytic leukemia (CLL) patients, isolated on a Ficoll-Hypaque gradient. We studied 18 CLL patients with median age of 65 y.o. (range, 45 to 86) out of treatment for at least three months (Rai 0 n=8; Rai 1 n=6; Rai 2 n=1; Rai3 n=1; Rai 4 n=2). As normal control we used 6 blood donors. Our data showed an overexpression of the human leukocyte formin in the CLL group when compared with the control group (p= 0.0354), as well as a positive correlation of this protein and ZAP-70 in the CLL group (Spearman test, p= 0.0107). The expression of ZAP-70 has been associated with rapid progression and poor survival and can be used as a prognostic marker. Previously we had described that human leukocyte formin protein associates with Akt, a critical survival regulator in many different cell types. The association was observed in a protein extract of Jurkat cell line and in peripheral blood leukocytes from CLL patients. In an attempt to confirm the association between Akt and human leukocyte formin, we performed cotransfections in 293 cells using an expression vector pEGFP containing FH2 or FH3 domains, and an expression vector of pCMV-HA containing the full length of Akt. The results showed that both FH2 and FH3 domains are involved in the association with Akt. The correlation of human leukocyte formin and ZAP-70 expression, and the association of human leukocyte formin protein with Akt suggest that this new protein may be involved in the signaling pathway of leukemia cell survival and is possibly a new prognostic marker.