ZAP-70 mRNA Research Articles

Normal B cells express ZAP70 in chronic lymphocytic leukemia: A link between autoimmunity and lymphoproliferation?

ZAP70 has a prognostic value in chronic lymphocytic leukemia (CLL), through altered B-cell receptor signaling, which is important in CLL pathogenesis. A good correlation between ZAP70 expression in CLL cells and the occurrence of autoimmune phenomena has been reported. Yet, the great majority of CLL-associated autoimmune cytopenia is due to polyclonal immunoglobulin (Ig) G synthesized by nonmalignant B cells, and this phenomenon is poorly understood. Here, we show, using flow cytometry, that a substantial percentage of CD5- nonmalignant B cells from CLL patients expresses ZAP70 compared with CD5- B cells from healthy subjects. This ZAP70 expression in normal B cells from CLL patients was also evidenced by the detection of ZAP70 mRNA at single-cell level with polyclonal Ig heavy- and light-chain gene transcripts. ZAP70+ normal B cells belong to various B-cell subsets and their presence in the naïve B-cell subset suggests that ZAP70 expression may occur during early B-cell development in CLL patients and potentially before malignant transformation. The presence of ZAP70+ normal B cells is associated with autoimmune cytopenia in CLL patients in our cohort of patients, and recombinant antibodies produced from these ZAP70+ nonmalignant B cells were frequently autoreactive including anti-platelet reactivity. These results provide a better understanding of the implication of ZAP70 in CLL leukemogenesis and the mechanisms of autoimmune complications of CLL.

A Percentage of Normal B Cells from CLL Patients Express ZAP-70, Which Is Associated with Autoimmunity and Could be Responsible for Arising of Malignant Clone

IntroductionChronic lymphocytic leukemia (CLL) has the particularity of a dysregulated immune system that favors the development of autoimmunity (AI). The mechanisms that lead to AI in CLL are not clear, but the great majority of pathogenic autoantibodies are polyclonal IgG synthesized by non-malignant B cells (Kipps and Carson 1993). ZAP-70 is the most discriminative marker between CLL patients with or without AI (Zanotti et al. 2010). We hypothesized that in CLL ZAP-70 expression is not restricted to malignant B cells, but could rather be an earlier phenomenon, predisposing to malignancy and AI.MethodsExpression of ZAP-70 in CLL, normal B and T cells was analyzed in 63 CLL patients and in 36 controls by flow cytometry and RT-qPCR. Then, single normal B cells were FACS-sorted, the mRNA was reverse-transcribed, and amplified for Ig heavy and light chain (IgH/L) as well as ZAP-70 genes. We cloned and expressed in vitro recombinant antibodies (rAb) from normal ZAP-70+ B cells and studied their reactivity in ELISA and on HEp-2 cells.ResultsWe clearly demonstrated that a percentage of normal B cells from CLL patients express ZAP-70, as compared to normal B cells from controls, at the protein level as shown by flow cytometry and at mRNA level by RT-qPCR experiments, although the later was not statistically significant (Fig 1A). This was definitely proven by sequence analysis of ZAP-70 amplified from single normal B cells mRNA (Fig 1B). These cells could not represent a contamination of normal B cells with a contingent of the CLL clone because they express a high level of CD19 and surface IgL and a normal k/λ ratio. The polyclonality of these cells was further confirmed by the analysis of IgH/L sequences rearranged variable regions (V) genes amplified from ZAP-70+ normal single B cells.ZAP-70+ normal B cells do not belong to a specific B-cell subset and do not express activation markers as compared with normal B cells from the same patient. Moreover, we observed a clear correlation between the expression levels of ZAP-70 in malignant, normal B and T cells in each CLL patient.Importantly, we found a percentage of ZAP-70+ normal B cells in all of the AI-associated CLL cases in our cohort. To study the role of these cells in the pathogenesis of autoimmunity, they were single-cell sorted and the IgH/L V genes as well as ZAP-70 were amplified by RT-qPCR. After sequencing of PCR products we found that in each patient a proportion of normal B cells indeed actively transcribe ZAP-70. This is the first time that ZAP-70 mRNA can be detected in single normal B cells, at the same time as IgH/L V transcripts.We cloned and expressed in vitro 13 rAb from normal ZAP-70+ IgD- class switched B cells and we found that 4 of the rAb reacted with HEp-2 cells and 2 were polyreactive (Fig 1C and D). This was not different from the class-switched normal B cells described in the literature (Tiller et al. 2007; Schickel et al. 2017), but the reactivity of more rAb is ongoing. Also, we plan to test them in MAIPA and Coombs tests to see if they could be at the origin of AI in CLL patients.We then compared IgH/L V genes sequenced from single cells between ZAP-70+ and ZAP-70- normal B cells and CLL B cells from 7 patients with CLL and AI. CDR3 from clonal CLL cells tend to be longer than those from normal B cells in line with the hypothesis that clonal CLL cells originate from autoreactive B cells which have undergone receptor editing (Gacia-Munoz et al. 2012), and VH replacement (Zhang et al. 2004). This is the case for one autoreactive ZAP-70+ normal B cell (HIT7) that expresses the same light chain as CLL clone, but a different and shorter IgH CDR3. In addition, some cells share the same IgH but display different IgL, therefore suggesting receptor editing.ConclusionsWe describe a novel normal B-cell population that expresses ZAP-70. There is a good correlation in the level of ZAP-70 expression between normal B cells and CLL B cells. The advent of AI in CLL is associated with the expression of ZAP-70 by clonal CLL cells as well as by normal residual B cells as we found a significant percentage of ZAP-70+ normal B cells in all AI-associated CLL cases. The reactivity of rAb produced from these cells is currently under evaluation. The presence of ZAP-70 in the naïve B-cell subset suggests that this is an early process, which probably occurs before malignant transformation. Analyzis of Ig sequences suggests a clonal relationship between ZAP-70+ normal B cells and CLL cells. [Display omitted] DisclosuresDrenou:Alexion: Consultancy, Honoraria.

Chemokine Receptors CCR1 and CCR2 on Peripheral Blood Mononuclear Cells of Newly Diagnosed Patients with the CD38-Positive Chronic Lymphocytic Leukemia

Chemokines and their receptors direct migration and infiltration of immune cells. CCR1 and CCR2 maintain sequence similarity and respond to a number of the same chemokines secreted in lymphoid organs. Expression of CD38 on leukemic cells has been associated with poor clinical outcomes in patients with chronic lymphocytic leukemia (CLL) and is considered as the negative predictor of progression. In our study of newly diagnosed CLL patients, which included 39 CD38-positive and 22 CD38-negative patients, CCR1 and/or CCR2 were always detected, using flow cytometry, on the peripheral blood (PB) CD19+CD5+ lymphocytes in patients with >30% of the CD38+ CD19+CD5+ lymphocytes (n = 16). Spearman’s rank correlation analysis determined correlations between the frequency of the CCR1- and CCR2-expressing PB CD19+CD5+ lymphocytes and the frequency of the CD38-positive CD19+CD5+ lymphocytes (rs = 0.50 and rs = 0.38, respectively). No significant correlations were observed between ZAP70 mRNA expression levels in PB mononuclear cells and the frequency of the circulating CCR1+ or CCR2+ CD19+CD5+ lymphocytes. Further association studies are needed to verify prognostic relevance of the CCR1/CCR2 expression on leukemic cells in CLL patients at diagnosis. We suggest that CCR1/CCR2 signaling pathways could represent attractive targets for development of CLL anti-progression therapeutics.

PD-1 expression on the surface of peripheral blood CD4+ T cell and its association with the prognosis of patients with diffuse large B-cell lymphoma.

The aim of the study was to investigate the relationship between PD‐1 expression on the surface of CD4+ T cells and prognosis of patients with diffuse large B‐cell lymphoma (DLBCL). Sixty patients who were newly diagnosed with DLBCL and 39 healthy controls were enrolled. In CD4+ T cells of DLBCL patients, the median MFI of PD‐1 were 541.5 (range: 348.25–758.75), significantly higher than 250 (range: 211–326) in healthy controls (P < 0.001). The ZAP70, PI3K, and NFAT mRNA expression levels of patients were 0.47, 0.47, and 0.62 times, respectively, of those of the healthy controls (P < 0.05). In patients with the percentage of PD‐1 on CD4+ T cells ≥30.25%, their EFS and OS were significantly lower than patients with PD‐1+ CD4+ T cells <30.25% (P < 0.05). The possible explanation is that high PD‐1 expression on CD4+ cells of DLBCL patients may impair T‐cell function and thus contribute to poor prognosis. There was no relationship between PD‐1 surface expression on CD4+ T cells and PD‐1 expression within the biopsy of tumor microenvironments from DLBCL patients.

Zeta-Chain-Associated Protein Kinase-70 (Zap-70) Promotes Acute Lymphoblastic Leukemia (ALL) Infiltration into the Central Nervous System By Enhancing Chemokine Receptor 7 (CCR7) Expression

Most children with B-cell precursor ALL (BCP-ALL) have an excellent prognosis but some patients are susceptible to CNS relapse, the mechanisms of which are largely unknown. Zap-70 is a tyrosine kinase mainly expressed in normal T- and NK-cells, but it is also expressed at low levels in normal B-cells. Zap-70 is however overexpressed in B-cell chronic lymphocytic leukemia (B-CLL) and is associated with an aggressive course of the disease. In B-CLL, Zap-70 was shown to up-regulate the expression of CCR7 (Calpe et al., 2011), which in turn allows T-lymphoblasts to enter the CNS (Buonamici et al., 2009). We hypothesized that Zap-70 mediated upregulation of CCR7 may enhance the homing and the survival of CNS-prone BCP-ALL cells in the CNS niche.We first transduced the 697 BCP-ALL cell line with a lentiviral vector carrying a non-targeting GFP (shGFP) or a Zap-70 (shZap-70) specific shRNA. Down-regulation of Zap-70 resulted in a significantly reduced CCR7 mRNA and surface protein expression. 697shGFP and 697shZap-70 cells were then injected intravenously into NSG mice, which were sacrificed when leukemic symptoms developed. There were no statistically significant differences in leukemic bone marrow infiltration and survival between the groups. However, immunohistochemical scoring of CNS infiltration (Krause et al., 2015) revealed that 9/10 animals (90%) in the 697shGFP group were CNS+ and only 2/7 animals (29%) in the 697shZap-70 group showed a CNS+ status (p = 0.0345). This suggests that Zap-70 mediated reduction in CCR7 impairs the CNS-positive phenotype of 697 cells in vivo.We next investigated if the Zap-70/CCR7 axis influences the ability of primary samples from patients to infiltrate the CNS of xenografts. Zap-70 mRNA was measured in pediatric BCP-ALL patients and patients with Zap-70 expression levels higher than the median were considered Zap-70high, and the remaining patients Zap-70low. 1 x 106 cells of 5 Zap-70high and 5 Zap-70low samples were injected intrafemorally into duplicate NSG mice. Interestingly, 7/10 (70%) of the mice injected with Zap-70high cells showed a CNS+ phenotype, whereas only 1/10 (10%) of the mice bearing Zap-70low samples were CNS+ (p = 0.0198). Importantly, leukemic cells of one Zap-70high patient purified from the xenograft CNS showed a marked upregulation of CCR7 expression as compared to cells isolated from bone marrow or spleen. These data suggest that the Zap-70/CCR7 axis is beneficial for patient BCP-ALL cells in the CNS of NSG mice.To test whether Zap-70/CCR7 is associated with CNS involvement in patients, we analyzed Zap-70 and CCR7 mRNA expression in diagnostic primary material of 76 BCP-ALL patients. The cohort contained 21 CNS-positive and 55 CNS-negative patients and was previously published by Cario et al. (2007). Patients were grouped into either Zap-70high/Zap-70low or CCR7high/CCR7low groups according to expression levels higher or lower than the median of the respective gene. Zap-70 expression was highly correlated with CCR7 expression (p = 0.0003; Spearman r = 0.401). There were no statistically significant differences in outcome surrogates such as prednisone response or MRD and there was no correlation between Zap-70/CCR7 expression and outcome (relapse or death). However, a trend towards high CCR7 levels and the occurrence of death could be observed (p = 0.055). Interestingly, high expression of Zap-70 or CCR7 correlated with the presence of blasts in the cytospin of the initial cerebrospinal fluid sample (p = 0.014 and p = 0.045, respectively). Furthermore, there was a significant correlation between high Zap-70 and a higher CNS status and a trend between high CCR7 and a higher CNS status according to AIEOP-BFM 2009 criteria (p = 0.024 and p = 0.098, respectively). We finally performed comparative correlations in 48/76 patients (63.2%) that were Zap-70high/CCR7high (n=24) or Zap-70low/CCR7low (n=24). Most importantly, a significant correlation between Zap-70high/CCR7high expression and a higher CNS status could be observed (p = 0.009). These data suggest that a high expression of Zap-70 or CCR7 is associated with CNS infiltration in BCP-ALL patients and that measurements of Zap-70 and CCR7 expression should be combined.Altogether, we show that Zap-70/CCR7 is important for BCP-ALL cells to enter the CNS and to survive in that niche. We propose Zap-70/CCR7 as a mechanism of CNS leukemia and a diagnostic marker with potential therapeutic implications. DisclosuresNo relevant conflicts of interest to declare.

ZAP70 Expression Within del6q21, del11q13 and del17p13 Cytogenetic Subgroups of Iranian Patients with Chronic Lymphocytic Leukemia.

Background:B-cell chronic lymphocytic leukemia (B-CLL) is the most common form of leukemia in adults. Some reports showed that expression of ZAP70 gene and chromosomal abnormality are two prognostic factors in management of B-CLLObjectives:In this study, we determined ZAP70 mRNA expression level in the del17p13, del6q21 and del11q13 subgroups of Iranian B-CLL patients to investigate prognostic value of ZAP70 expression.Patients and Methods:In this cross-sectional study, fluorescence in situ hybridization analysis was carried out on 66 Iranian B-CLL patients. Zap70 mRNA expression was evaluated by using Real Time RT-PCR.Results:Molecular analysis showed that ZAP70 expression increased 2.46 fold in the del11q13 subgroup, 2.87 fold in the del17p13 and 1.87 fold in the del6q21, compared to the 15 patients in the control group. Comparison of standard deviation and mean of the ZAP70 expression profile within the subgroups showed more variability among the cases of the del11q13 and del17p13 versus tight clustering for the del6q21. Therefore, there is a relation between del6q21 aberrations; which has good prognosis with normal levels of ZAP70 expression.Conclusions:The results of ANOVA test showed that ZAP70 expression gene was significantly increased in del17p13 and del11q13 subgroups compared to control group. Thus, ZAP70 may play an important role in the prognosis of B-CLL patients.

ZAP70 Methylation Is An Independent Prognostic Biomarker For Front Line Therapy Of Chronic Lymphocytic Leukemia : Results From The UK LRF CLL4 Trial

In CLL, over-expression of ZAP70 is well established as a prognostic biomarker for unfavourable disease course. However, technical challenges limit the application of flow cytometry for ZAP70 detection and measurement of ZAP70 mRNA expression is complicated by the need for T cell depletion. Having identified a correlation between DNA methylation of a 5' regulatory region of ZAP70 and expression of the gene, we and others have proposed the measurement of ZAP70 methylation as an alternative biomarker. Of note, recent work by Claus et al showed ZAP70 methylation to be prognostic for progression free survival (PFS) after therapy in two small, phase II, trial cohorts, treated with regimes incorporating Rituximab. However, as no comprehensive assessment of the prognostic value of ZAP70 methylation has been reported, we analysed ZAP70 methylation in 416 samples from patients entered into the phase III UK LRF CLL4 trial (comparing Chlorambucil(C) with Fludarabine alone (F) or in combination with Cyclophosphamide(FC)).The methylation of a single CpG site 334bp downstream of the transcription start site (C334) was interrogated in peripheral blood mononuclear cell DNA samples, using a quantitative bisulfite pyrosequencing assay. All samples were bisulfite converted, amplified and pyrosequenced in triplicate with a 6 point standard curve in each run. Standard curves for each of the 20 runs were linear (r2 > 0.98) and sensitivity of pyrosequencing was ±5%.C334 methylation ranged from 0-100% and showed a significant inverse correlation with percentage of ZAP70 positive cells, IGHV identity to germline and percentage of CD38 positive cells (all p≤0.001). Methylation levels showed a trimodal, W-shaped, distribution with major modes at 5% and 95% and a minor mode at 50%. Based on this distribution, samples were classified as having Low (C334L<33.3%), Intermediate (C334Int 33.3-66.6%) or High (C334H >66.6%) methylation (Figure 1). [Display omitted] In univariate Kaplan Meier (KM) analysis, no significant difference was seen between C334L and C334Int groups in terms of PFS or OS (median PFS 1.68 versus 1.76 years, p=0.36 and median OS 4.71 years versus 5.41 years, p=0.18). However, C334H cases had a significantly longer PFS and OS than either group (median PFS=2.86 years, median OS=8.60 years, all p<0.001). C334H cases were significantly enriched with good risk features such as the presence of a 13q deletion, absence of 11q deletion, absence of NOTCH1 mutation, ZAP70 negativity, CD38 negativity and IGHV mutated status (all p<0.02). However, C334H status retained significant prognostic value for PFS and OS in KM analysis when cases were stratified by each of these good risk features. Furthermore, in multivariate Cox regression analysis, C334H cases showed a significantly reduced risk of progression compared to all other cases (HR=0.67 (95%CI 0.48-0.95), p=0.023), independent of treatment received, IGHV status and 11q and 17p deletion status (Table 1).By identifying a group of patients with hypermethylation of ZAP70, who display a superior outcome with first line chemotherapy, our results confirm the prognostic value of ZAP70 methylation and show it to be superior to ZAP70 expression as measured by flow cytometry. The independent prognostic value of this, compared with established biomarkers, together with the ease and low cost of the assay, warrant its validation in other large trial cohorts. Disclosures:No relevant conflicts of interest to declare.

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

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. 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. The expression of ZAP-70 mRNA was significantly associated with Binet stage (P < 0.001), lactate dehydrogenase (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. 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.

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.

Analysis of CD38 and ZAP70 mRNA expression among cytogenetic subgroups of Iranian chronic-lymphocytic-leukemia patients

Chromosomal abnormalities and ZAP70 expression profile are two major independent prognostic markers in B-cell chronic lymphocytic leukemia. We investigated a possible correlation between these two markers. ZAP70 expression using real-time RT-PCR was examined in 20 B-cell chronic lymphocytic leukemia patients with del13q14, 13 patients with del11q22, 15 patients with trisomy 12, and 16 patients with no detected chromosomal abnormalities. Molecular analysis revealed that ZAP70 expression in the del13q subgroup was the same as in the control group, while it increased 2.78-fold in the del11q subgroup and 2.95-fold in the trisomy 12 subgroup, compared to the 15 cases in the control group. Comparison of the mean and standard deviation of the ZAP70 expression profile within the subgroups showed it to be highly variable among the individuals of the del11q and trisomy 12 subgroups, versus tight clustering for the del13q subgroup. Therefore, there is a correlation between del13q aberration, which has good prognosis with normal levels of ZAP70 expression. Due to a high degree of variation, no conformity is seen for del11q and trisomy 12 subgroups, making this grouping poor for prognostic discrimination. As a result, neither of these markers can serve as sole discriminators to determine the course of the disease; the use of both markers improves prognostic assessment.

Quantification of ZAP70 mRNA in B Cells by Real-Time PCR Is a Powerful Prognostic Factor in Chronic Lymphocytic Leukemia

Quantification of ZAP70 mRNA in B Cells by Real-Time PCR Is a Powerful Prognostic Factor in Chronic Lymphocytic Leukemia

ZAP-70 and Syk expression in canine lymphoid cells and preliminary results on leukaemia cases

Zeta-chain-associated protein (ZAP-70) and spleen tyrosine kinase (Syk) are structurally and functionally homologous tyrosine kinases playing a role in the T- and B-cell signal transduction. Their activation can lead to lymphokine production, cytolitic activity, antibody secretion, cell proliferation, differentiation, survival and phagocytosis. Anomalous ZAP-70 and Syk expression is reported to be related to tumor formation and progression, and ZAP-70 immunoreactivity is a good prognostic marker of disease progression in human chronic lymphocytic leukaemia (CLL). Until now, to our knowledge, there are no reports about canine ZAP-70 and Syk expression profiles. In the present study, a RT-PCR procedure for the quali-quantitative evaluation of canine ZAP-70 and Syk transcripts was designed. The expression patterns of canine ZAP-70 and Syk mRNAs were evaluated in canine leukocyte subpopulations and in peripheral whole blood samples from healthy dogs and from dogs with different types of leukaemia. Similarly to humans, normal canine CD4+ and CD8+ T cells showed high expression of ZAP-70, whereas Syk was abundantly expressed in normal CD21+ B cells. The expression profile of ZAP-70 and Syk was markedly different in canine normal and leukaemic blood. Decreased Syk expression was detected in dogs with T-cell CLL, whereas decreased ZAP-70 expression was detected in dogs with B-cell CLL and B-cell acute lymphocytic leukaemia (ALL). The comparison of ZAP-70 and Syk mRNA levels between normal and leukaemic peripheral whole blood showed that the expression ratio ZAP-70/Syk is subjected to modification depending on the leukaemia status of patients. The results of the present work open an interesting topic for leukaemogenesis investigation and are the basis for further studies for a proper evaluation of the potential utility of these parameters for the diagnosis and prognosis of canine T- and B-cell leukaemia.

Expression profile of Eph receptors and ephrin ligands in healthy human B lymphocytes and chronic lymphocytic leukemia B-cells

Increasing information relates some Eph receptors and their ligands, ephrins (EFN), with the immune system. Herein, we found that normal B-cells from peripheral blood (PB) and lymph nodes (LN) showed a differential expression of certain Eph/EFN members, some of them being modulated upon in vitro stimulation including EFNA1, EFNA4, EphB6 and EphA10. In contrast, PB CLL B-cells showed a more heterogeneous Eph/EFN profile than their normal PB B-cell counterparts, expressing Eph/EFN members frequently found within the LN and activated B-cells, specially EFNA4, EphB6 and EphA10. Two of them, EphB6 and EFNA4 were further related with the clinical course of CLL patients. EphB6 expression correlated with a high content of ZAP-70 mRNA and a poor prognosis. High serum levels of a soluble EFNA4 isoform positively correlated with increasing peripheral blood lymphocyte counts and lymphadenopathy. These findings suggest that Eph/EFN might be relevant in normal B-cell biology and could represent new potential prognostic markers and therapeutic targets for CLL.

ZAP-70 mRNA expression provides clinically valuable information in early-stage chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable clinical course and overall survival times ranging from months to decades.[1][1] Mutational status of immunoglobulin heavy chain variable region (IGHV) correlates with clinical behavior and is a powerful prognostic factor

Apoptosis in Response to Inhibition of Dipeptidyl Peptidase 2 (DPP2) Defines Low Risk Chronic Lymphocytic Leukemia (CLL).

Maintenance of the G0 state is a key to survival of CLL B-cells. Heterogeneity of prognosis suggests that CLL is not a uniform disease. Molecules expressed in CLL with unfavorable prognosis, such as ZAP-70, Lyn, CD38 and others, provide stimuli which, coupled with B-cell receptor signaling, may alter cell cycle progression and delay apoptosis. We studied mechanisms of apoptosis in CLL B-cells via inhibition of Dipeptidyl Peptidase 2 (DPP2). DPP2 is a serine protease cloned in our lab, which is involved in the maintenance of the G0 (quiescent) state. Inhibition of DPP2 triggers apoptosis in healthy B-cells. 50 patients with B-CLL were recruited from the Hematology clinics at Tufts-NEMC (Boston, MA). Median time from diagnosis of CLL to enrollment in the study was 7 years with median follow up of 10.5 years. 24 patients (48%) have received treatment in the course of their disease. CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique, treated with ValboroPro (Point Therapeutics), a non-specific inhibitor of DPPs, and/or AX8819 (ActivX), a DPP2-specific inhibitor, incubated for 16 hours and stained with anti-CD19 antibodies, propidium iodide and Annexin V. Expression of DPP2 and ZAP-70 mRNA was assessed by RT-qPCR, p27 protein - by western blot analysis. By blocking DPP2 protease activity, we distinguished two subsets of CLL - sensitive (S-CLL) and resistant (R-CLL) to DP-P2 inhibition-induced apoptosis. In 30 cases (60%), inhibition of DPP2 resulted in caspase-dependent apoptosis of CLL B-cells (S-CLL). 70–90% of B-cells stained Annexin V-positive after incubation with AX8819. Pre-incubation with Rituximab (Biogen Idec) did not enhance cell death. In the remaining 20 cases (40%) inhibition of DPP2 did not cause cell death (R-CLL). R-CLL demonstrated higher expression of ZAP-70 mRNA (p<0.001) and lower levels of p27 protein (p<0.01) than S-CLL. ZAP-70 mRNA levels strongly correlated with resistance to apoptosis (χ2=26.7, p<0.0001). Inhibition of DPP2 resulted in a decrease in p27 protein levels in S-CLL, but not in R-CLL. Both groups expressed higher DPP2 mRNA levels than B-cells derived from healthy donors. In the R-CLL subgroup, 18 patients (84%) required treatment for their disease, 14 received more than one treatment regimen, 2 underwent allogeneic transplant and 2 died of complications of CLL. Among the S-CLL cohort, 10 patients (33%) initiated treatment, 4 received more than one treatment regimen. R-CLL cohort required treatment earlier than S-CLL (3.5 vs. 9.9 years from diagnosis). Thus, DPP2 inhibition discriminates two subsets of CLL based on their ability to undergo apoptosis upon disruption of the quiescent program. We postulate that DPP2 is critical for the maintenance of G0 in S-CLL. Its inhibition leads to inappropriate cell cycle entry, as evidenced by a decrease in p27 protein levels and cell death. The R-CLL subgroup expresses high ZAP-70 mRNA levels which portends a worse clinical course. In this subgroup, ZAP-70 kinase activity may provide an additional tonic signal that pushes CLL B-cells into late G0/early G1. At this stage, DPP2 protease activity is no longer required for survival. DPP2 inhibition may serve as an easy-to-perform prognostic test, as well as a novel therapeutic approach.

Flow Cytometric Determination of ZAP-70 Expression by SBZAP Antibody: Impact of Type of Data Analysis on Correlation with IgVH Mutation Status, Cytogenetics, and Level of ZAP-70 mRNA Expression in 351 Patients with Chronic Lymphocytic Leukemia (CLL).

ZAP-70 is increasingly used for prognostication in CLL. However, type of data analysis and most appropriate antibody clones need to be defined. In 351 patients (pts) we analyzed ZAP-70 expression flow cytometrically using both SBZAP (Immunotech) and 1E7.2 (Caltag) antibody clones in parallel (both PE-conjugated). In 260 pts the SBZAP kit was used (combined with CD19/CD5/CD3) as specified by the manufacturer (referred to as “SBZAPkit”). In 104 pts (incl. 13 of previous 260) SBZAP was used with a different antibody combination (TdT/cCD79a/cCD3; custom design service (CDS) program, Immunotech; “SBZAPcds”). 1E7.2 was used in all pts. Data analysis for ZAP-70 expression was done in 3 different ways: % positive leukemic cells using normal T-cells as positive controls, mean fluorescence intensity (MFI) in leukemic cells, MFI-ratio between T- and leukemic cells. IgVH mutation status was analyzed in 226 pts and chromosome aberrations in 223 pts applying FISH with probes for del(6q), del(11q), +12, del(13q), del(17p), and t(11;14). % ZAP-70+ cells correlated with % IgVH homology (SBZAPkit, p&lt;0.0001, r=0.358; SBZAPcds, p=0.003, r=0.403; 1E7.2, p=0.103, r=0.111). Accordingly, positivity for ZAP-70 (&gt;20% positive cells, “Z+”) correlated with unmutated IgVH status (≥98% homology, “VHu”) although discordant cases frequently occured; concordant results were obtained in 71.4% (SBZAPkit, p&lt;0.0001), 69.8% (SBZAPcds, p=0.004), and 47.0% (1E7.2, p=0.03). Better correlations with IgVH mutation status were found for ZAP-70 MFI in leukemic cells: SBZAPkit, p&lt;0.0001, r=0.462; SBZAPcds, p&lt;0.0001, r=0.566; 1E7.2, p&lt;0.0001, r=0.296. Accordingly, a better concordance was observed between ZAP-70 positivity (MFI cut-offs: SBZAPkit, 1.5; SBZAPcds, 0.75; 1E7.2, 1.0) and IgVH mutation status: SBZAPkit, 75.2%, p&lt;0.0001; SBZAPcds, 84.9%, p&lt;0.0001; 1E7.2, 75.3%, p&lt;0.0001. Mean MFI-values for VHu v VHm cases amounted to 1.29 v 0.73 (SBZAPkit, p&lt;0.0001), 1.12 v 0.46 (SBZAPcds, p&lt;0.0001), and 0.96 v 0.75 (1E7.2, p&lt;0.0001). Evaluating ZAP-70 expression as MFI-ratio T:B-cells also resulted in strong correlations with IgVH mutation status for SBZAP: SBZAPkit, p&lt;0.0001, r=−0.471; SBZAPcds, p=0.003, r=−0.402; 1E7.2, p=0.023, r=−0.153. Accordingly, a high degree of concordance was observed between ZAP-70 positivity (MFI cut-offs: SBZAPkit, 4.5; SBZAPcds, 4.5; 1E7.2, 3.0; rated positive if falling below cut-off) and unmutated IgVH status: SBZAPkit, 74.2%, p&lt;0.0001; SBZAPcds, 66.5%, p=0.008; 1E7.2, 43.9%, p=0.162. Mean MFI-ratios for VHu v VHm cases were 3.35 v 6.08 (SBZAPkit, p&lt;0.0001), 3.59 v 6.32 (SBZAPcds, p=0.01), and 2.14 v 2.73 (1E7.2, p=0.02). A high degree of correlation with levels of ZAP-70 mRNA expression as determined by quantitative PCR (n=44) was found for both MFI (p=0.002, r=0.461) and MFI-ratio (p&lt;0.0001, r=0.596) using SBZAPkit. In pts rated ZAP-70 positive v negative by MFI-ratio mean ZAP-70 mRNA levels were 0.41 v 0.04 (p=0.002). No consistent associations of ZAP-70 expression assessed by flow with chromosomal aberrations was observed. The present results confirm the strong correlation between ZAP-70 expression and IgVH mutation status and its independence of chromosome aberrations. The data favor the evaluation of ZAP-70 expression using MFI in leukemic cells and the MFI-ratio between T-cells and leukemic cells over percentages of positive cells. SBZAP carries a significant potential for the diagnostic work-up in CLL.

T Cell Pathway Deficiencies in Chronic Lymphocityc Leukemia: Partial Restoration with OKT3/IL-2 Activation.

As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p<0.05) in CD3+ cells from CLL patients compared with healthy donors. Panther software analysis identified 34/237 upregulated genes and 26/221 downregulated genes that were involved in specific pathways, mainly cell differentiation and proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T cell activation. The 26 dowregulated genes included Zap70, a member of the syk family protein tyrosine kinase, which is involved in T-cell activation. Zap-70 results were validated by mRNA quantification by RT-PCR (−1.77 fold in comparison with healthy controls) and by flow-cytometric analysis (Mean Intensity Fluorescence=33±12 vs 80±23.62 in controls, p<0.05). To test the hypothesis that activation with OKT3 /IL-2 could bypass these T cell deficiencies, activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity (using the 51chromium release assay) against mutated and unmutated (according to IgVH mutational status) autologous B cells, DAUDI, K562 and P815 cell lines. After 10 days' culture, the T cell count remained unchanged; CD8 cells expanded more than CD4; TCR spectratyping analysis indicated no differences in TCR repertoires. Activation restored the ZAP-70 mRNA (+1.67 fold). The 51chromium release cytotoxicity assay showed an index > 30% in 5/20 patients. The other 15 were partially cytotoxic against P815, K562 and Daudi. Cell line analysis in all 20 confirmed prevalently T cell-mediated cytotoxicity and poor NK/LAK activity. Cytotoxicity did not correlate with B cell mutational status. We tested the cytotoxic activity of autologous activated T cells in NOD/SCID mice co-transplanted with leukaemic B cells. Only activated T cells exerting cytotoxicity vs autologous B-cell CLL prevent CLL in human-mouse chimera, as confirmed by PCR and FACS analysis which visualised only CD3+ cells. In conclusion, in patients with CLL, activating autologous T cells with OKT3 /IL-2 bypasses, at least in part, the T cell immunological deficiencies. These in vitro and in vivo findings might serve to throw light on new mechanisms that could be exploited in immunotherapy designed to exert disease control.

Efficient gene transfer in CLL by mRNA electroporation

Chronic lymphocytic leukemia (CLL) consists of at least two major prognostic subgroups, characterized by different cellular and molecular markers. This observation sparked studies on the function and clinical importance of these markers. In order to address their function adequately, an efficient and reliable method for gene transfer is needed. In this study, we compared efficiency and utility of different gene transfer techniques in CLL. Lenti-, retro- and adenoviral transduction did not yield appreciable numbers of marker gene enhanced green fluorescent protein (EGFP) positive CLL cells, despite various prestimulation protocols. Efficient transgene expression was observed after nucleofection of CLL cells with plasmid DNA, at the expense of low survival rates. After optimization, electroporation of in vitro transcribed mRNA yielded up to 90% EGFP+CLL cells without affecting survival. Transgene expression remained detectable for at least 2 weeks after electroporation. Furthermore, we could demonstrate overexpression of ZAP70 and of a ZAP70-EGFP fusion protein after electroporation with ZAP70 or ZAP70-EGFP mRNA. We conclude that mRNA electroporation is a novel and straightforward method for highly efficient gene transfer in CLL. The application of this technique should facilitate functional studies on CLL cells, as well as clinical research.

Quantification of ZAP70 mRNA in B Cells by Real-Time PCR Is a Powerful Prognostic Factor in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is heterogeneous with respect to prognosis and clinical outcome. The mutational status of the immunoglobulin variable heavy chain region (IGHV) has been used to classify patients into 2 groups in terms of overall survival (OS) and clinical characteristics, but the labor-intensive nature and the cost of this time-consuming analysis has prompted investigations of surrogate markers. We developed a standardized quantitative real-time reverse transcription-PCR (qPCR) method to measure zeta-chain (TCR)-associated protein kinase (ZAP70) mRNA in purified CD19(+) cells. We evaluated this and other methods (flow cytometry analyses of ZAP70 and CD38 proteins and qPCR analysis of lipoprotein lipase mRNA) in a cohort of 108 patients (median follow-up, 82 months) to evaluate any associations with IGHV mutational status, OS, and treatment-free survival (TFS). The association between qPCR-measured ZAP70 and IGHV mutational status was statistically significant [chi(2) (1) = 50.95; P <0.0001], and the value of Cramer's V statistic (0.72) indicated a very strong relation. This method also demonstrated sensitivity, specificity, and positive and negative predictive values of 87.8%, 85.7%, 87.5%, and 86%, respectively. ZAP70 expression was significantly associated with OS (P = 0.0021) and TFS (P <0.0001). ZAP70(+) patients had significantly shorter median TFS (24 months) than ZAP70(-) patients (157 months) (P <0.0001). Moreover, qPCR-measured ZAP70 expression has greater prognostic power than IGHV mutational status and the other prognostic markers tested. ZAP70 mRNA quantification via qPCR is a strong surrogate marker of IGHV mutational status and a powerful prognostic factor.

Novel mouse mutants with primary cellular immunodeficiencies generated by genome-wide mutagenesis

Primary cellular immunodeficiencies are a group of genetic disorders in which 1 or more components of the cellular immune system are lacking or dysfunctional. We sought to identify novel mouse mutants that display primary cellular immunodeficiencies. Genome-wide N-ethyl-N-nitrosourea mutagenesis was performed in mice, followed by a phenotype screen of immunologic blood parameters. We identified novel mouse mutants with isolated B-cell deficiency, combined block in early B- and T-cell development, combined T-cell and natural killer cell reduction, and 3 different forms of T-cell deficiencies. One of the mutants, designated DeltaT3, displayed a combined phenotype of increased IgE, absence of peripheral T cells, and block in late thymocyte differentiation. In addition, DeltaT3 mice were unable to mount specific humoral immune responses. Chromosomal mapping and sequencing of candidate genes revealed a novel point mutation in the kinase domain of the T-cell receptor zeta chain-associated protein kinase (Zap70). In contrast to Zap70-deficient mice, DeltaT3 mutants displayed normal Zap70 mRNA and residual Zap70 protein levels. Complementation studies with Zap70-deficient mice confirmed that the point mutation found in Zap70 was causative for the DeltaT3 phenotype, including increased IgE plasma levels, a phenotype that has not been associated with altered Zap70 function in the past. Random genome-wide mutagenesis combined with a phenotype screen can be used to generate novel mouse mutants with primary cellular immunodeficiencies.