ZAP-70 Positive Chronic Lymphocytic Leukemia Research Articles

ZAP-70 Integrates Tonic B Cell Receptor and Chemokine Signaling in IGHV unmutated Chronic Lymphocytic Leukemia

The importance of B cell receptor (BCR) signaling for the progression of Chronic Lymphocytic Leukemia (CLL) has impressively been demonstrated through the clinical success of small molecule inhibitors interfering with this pathway, providing effective treatment options for a vast majority of patients. The scientific evidence which supported the development of drugs like BTK and PI3K inhibitors largely originate from studies of antigen-induced BCR-signaling. Notably, the presence of a constitutive, so-called tonic BCR-signal was long postulated to co-exist besides antigen-driven activation and is based on the identification of constitutively activated signaling molecules down-stream of the BCR. We here provide direct evidence for the presence of a tonic BCR-signal, which critically relies on the expression of the tyrosine kinase ZAP-70. We have previously established a method to inhibit ZAP-70 expression in primary CLL cells usingRNA-interference in combination with an extended co-culture system. Our previous work demonstrated that ZAP-70 is required for the constitutive gene expression of T cell chemokines CCL3 and CCL4 and contributes to the dysregulation of global protein synthesis and MYC expression in CLL ( Blood, 2021). Applying this method, we here provide evidence for the presence of a constitutive tonic BCR signal, which is dependent on ZAP-70 expression and exclusively found in IGHV unmutated CLL, but not in IGHV mutated cases discordantly expressing ZAP-70. We demonstrate that ZAP-70 is directly involved in the constitutive activation of AKT and GSK-3beta. Furthermore, ZAP-70 expression increases steady state activation of CD19 and the downstream kinase SRC, providing direct evidence for the existence of a ZAP-70 dependent tonic BCR signal in I GHV unmutated CLL only. To identify the underlying signaling events driving this signal, we performed mass spectrometry in combination with proximity ligation assays on CLL cells in the absence of an induced BCR-signal. Unexpectedly, we identified that the majority of ZAP-70 binding proteins do not related to BCR-signaling components, but to cytoskeleton organizing proteins involved in cell migration. Indeed, migration assays demonstrated that the loss of ZAP-70 directly and significantly impaired cell migration induced by CCL19 and CCL21, in keeping with previous data indication an association between ZAP-70 and cell migration. These results were confirmed using a switchable model of ZAP-70, which allowed us to toggle ZAP-70 expression on and off at any given time. To further define the mechanisms of ZAP-70 mediated cell migration, we studied CCL19 and CCL21 signaling in ZAP-70 proficient and deficient cells. Unexpectedly, we identified that ZAP-70 is required for chemokine-mediated activation of AKT. In summary, we here demonstrate that a tonic BCR-signal is present only in IGHV-unmutated CLL and dependent on the expression of ZAP-70. Furthermore, ZAP-70 converges two seemingly independent signaling events, namely chemokine- and tonic BCR signaling. Our data provide further explanations for the more aggressive clinical course of ZAP-70 positive CLL.

BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies.

Study of microRNA Profile as a Molecular Biomarker in Egyptian Chronic Lymphocytic Leukemia.

MicroRNAs target mRNAs for cleavage or translational repression. They play a critical role in the progression of malignancies and leukemias including chronic lymphocytic leukemia (CLL). However, microRNA expression levels in Egyptian patients with CLL, and their prognostic value remain elusive. Our main aim was to assess the expression pattern of a panel of microRNAs in CLL patients to create an informative microRNA profile. The study subjects were 40 newly diagnosed CLL patients of both sexes and 40 age and sex matched controls. The expression levels of 12 microRNAs were evaluated by qRT-PCR, including miR-15a, 16, 23b, 24, 29a, 29c, 34a, 146a, 155, 181a, 195, and 221. Flow cytometry was used to determine the expression levels of BCL2, CD38, and ZAP-70 in CLL patients. We identified various degrees of upregulated miRNAs (miR-29a, miR-29c, miR-34a, miR-155, miR-146a, and miR-195) and down-regulated ones (miR-15a, miR-16, miR-23b, miR-24, miR-181a, and miR-221) in CLL patients relative to controls. The mean fluorescence intensity ratio (MFI-R) of BCL2 was recorded and was significantly upregulated in CLL patients compared with normal controls. In addition, inverse correlations were observed between microRNAs (miR-15a, miR-16, miR-155, and miR-195) and BCL2 MFI-R while positive correlations were observed between miR-29a and miR-29c, and BCL2 MFI-R. These findings suggest that these miRNAs regulate BCL2 levels. Moreover, we found that miR-15a, miR-16, miR-155, miR-181a, miR-195 and miR-221 were significantly upregulated, while miR-29a and miR-29c were significantly downregulated in ZAP-70 positive CLL patients. Various miRNAs may play an important role in the pathogenesis of CLL and have the potential to be used for the prognosis of patients with CLL.

Integration of innate into adaptive immune responses in ZAP-70–positive chronic lymphocytic leukemia

AbstractThe crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70–positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70–positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70–positive CLL.

IgM and IgD Receptors Differentially Contribute to CLL Survival and Chemokine Secretion: Implications for CLL Biology and Treatment

Background: B-cell receptor (BCR) signaling is a central pathway activated in the tissue microenvironment of patients with Chronic Lymphocytic Leukemia (CLL). CLL cells express IgM and IgD receptors, but the contribution of these isoforms to signaling and functional responses remains incompletely defined. The mutational status of CLL BCRs, on the other hand, has clear correlation with clinical outcome: patients with unmutated IGHV genes (U-CLL) characteristically have more aggressive disease and an inferior prognosis when compared to patients with mutated IGHV (M-CLL).Aim: The aim of this present study is to characterize functional differences between IgM and IgD signaling, in the context of the CLL microenvironment and of ibrutinib treatment.Methods: Freshly isolated, purified CLL cells from 80 patients (40 M-CLL and 40 U-CLL) were stimulated with soluble or bead-bound anti-IgM or anti-IgD; IgM, IgD expression, phosphorylation of the BCR-related molecules HS1 and ERK, F-actin polymerization and cell viability were analyzed by flow cytometry; HS1 and ERK phosphorylation, and BCL6 protein expression were also analyzed by Western Blot. CCL3 and CCL4 chemokine secretion was measured by ELISA. BCL6 binding to the CCL3 gene promoter region was assessed by ChIP-PCR. We utilized nurselike cell (NLC) co-cultures to activate BCR signaling and to induce CCL3 and CCL4 production in this model system that resembles the lymph node microenvironment. Effects of ibrutinib treatment on CLL-cell viability and BCR activation were also tested.Results: Surface IgM and IgD levels were significantly higher in U-CLL (P<0.0001), ZAP70 positive (P<0.01), and/or del17p or del11q CLL cells (P<0.05). Early BCR signaling with activation of the cytoskeletal protein HS1 and F-actin polymerization were similar after anti-IgM and anti-IgD stimulation. Downstream activation of ERK kinases was more prolonged after anti-IgM than after anti-IgD stimulation. Transient activation of IgD signaling was paralleled by rapid IgD receptor endocytosis, as compared to IgM. Furthermore, anti-IgM increased CLL viability to a significantly greater extent than anti-IgD, and exclusively induced secretion of CCL3 and CCL4 chemokines. This effect was consistently observed after stimulation with either soluble or bead-bound anti-IgM and anti-IgD. Expression of BCL6, which functions as a transcriptional repressor for CCL3 gene, was down-regulated following anti-IgM, but not after anti-IgD stimulation. In addition, we observed BCL6 binding to CCL3 promoter regions and noted that BCL6 down-regulation coincided with the onset of CCL3 secretion. In all experimental settings, U-CLL cells were more responsive to BCR stimulation than M-CLL. NLC co-culture induced down-modulation of surface IgM and IgD on CLL cells, increased HS1 and ERK phosphorylation and induced CCL3 chemokine secretion, along with BCL6 down-regulation. When CLL cells were removed from NLCs, recovery of surface IgM and IgD, baseline BCL6 expression, and HS1 and ERK phosphorylation was observed after 72 hours. Ibrutinib treatment preferentially interfered with anti-IgM induced survival signals and BCR signaling activation, in particular of U-CLL cells.Conclusion: Compared to IgD, IgM stimulation induces more durable signaling responses, CCL3 and CCL4 chemokine secretion and increases CLL-cell survival, in particular in U-CLL cells. BCL6 binding to CCL3 promoter and its down-regulation concomitant to CCL3 production suggests that BCL6 represses CCL3 gene expression in CLL cells. Co-culture of CLL cells with NLCs triggers BCR signaling, with functional outcomes that mainly recapitulate the features of IgM stimulation in vitro. Ibrutinib treatment preferentially interferes with IgM-mediated signals, and reduces CCL3 and CCL4 levels in CLL-cell supernatants and in the plasma of ibrutinib-treated patients. Taken together, these results support the relevance of IgM signaling in the pathogenesis of CLL, and suggest that IgM is the main receptor isotype targeted in vivo during ibrutinib treatment. DisclosuresEstrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Splicing Factor 3B Subunit 1 (SF3B1) Mutations Impact FOXP1 Splicing in Chronic Lymphocytic Leukemia (CLL) Cells but Conserve the Balance Between Newly Described and Classical Splicing Variants of ZAP-70

Zeta-chain Associated Protein of 70 kDa (ZAP-70) is the second member of the Spleen tyrosine Kinase (Syk) family, and plays a key role in T-cell Receptor signalling. An alternative variant of ZAP-70 was described in 2004 by Hirokawa et al. producing a shorter protein named Truncated ZAP-70 Kinase (TZK). ZAP-70 has also been found expressed in some B-cell subsets and in various B-cell malignancies. In CLL, the expression of ZAP-70 retains a prognostic significance; however, its function is not completely understood. Recently, whole genome sequencing identified mutations affecting the spliceosome such as SF3B1 mutations that are associated with poor outcome in CLL. In this work, we aimed at identifying novel alternative transcripts of ZAP-70 in normal lymphocytes and in CLL cells. We also explored the potential link between ZAP70 alternative transcripts and SF3B1 mutations.Peripheral blood mononuclear cells (PBMC) were obtained from normal subjects and CLL patients. T cells, NK cells and monocytes were purified by negative selection from 11 normal subjects. We explored the transcriptional profile of ZAP-70 by standard PCR using 4 combinations of primers spanning over the 14 exons of the transcript. PCR products were revealed by gel electrophoresis, purified and sequenced by Sanger method. The expression of ZAP-70 was studied by real-time quantitative PCR using SYBR Green® technique after designing specific primers for the four different transcripts individualized. The expression of the full length and truncated forms of FOXP1 was studied by real-time quantitative PCR as previously described (EHA2015 abst#4919). SF3B1 mutations were detected by Sanger sequencing of the exons 14, 15, and 16.In Jurkat cell line, we identified two new variants of ZAP-70 corresponding to the alternative splicing of exon 3 and exons 3 and 4. We named them Δ3 and Δ3-4 respectively after confirmation by Sanger sequencing. To detect both Δ3 and Δ3-4 in the same reaction, we designed a PCR encompassing exons 1 to 5, and we were able to detect ZAP-70, Δ3 and Δ3-4 in peripheral blood unselected normal cells. SYBR Green ® technique with new sets of primers allowed us to explore ZAP-70, TZK and the two new variants in selected NK cells and T lymphocytes. As previously described, ZAP-70 was more expressed in NK cells than in T cells, and at lower level in CLL cells. TZK was also detected in T-lymphocytes and NK cells, Δ3 and Δ3-4 were found at very low levels in both cell types and were absent from ZAP-70 negative CLL cells. We studied ZAP-70 transcripts in 13 ZAP-70 positive CLL. All alternative transcripts were present with a pattern of expression different to that of T-cells. We have shown recently that in all cases of CLL with SF3B1 mutation, a high expression of the FOXP1 truncated variant was observed as compared to a very low level in unmutated samples. This variant was highly expressed in the samples carrying five different mutations of SF3B1 (Y623C, R625H, K700E, G740V and G742D). Among our 13 ZAP70 positive patients, 2 cases harboured the SF3B1 Y623C mutation. In these two cases, the balance between the four ZAP-70 transcripts detected showed no obvious difference with wild-type SF3B1 ZAP-70 positive cases.In conclusion, we studied the transcription profile of ZAP-70 in various cells types and identified two novel alternative transcripts of ZAP-70 (Δ3 and Δ3-4). These transcripts were detected at various levels in all ZAP-70-positive cell types. Interestingly, we found that the presence of SF3B1 mutation impacted the splicing of FOXP1 transcription factor but not the balance between ZAP-70 transcripts We are currently expanding the cohort of ZAP-70 cases with other types of SF3B1 mutations. Mutations affecting the spliceosome such as SF3B1 mutations do not seem to impact transcription of ZAP-70. DisclosuresTroussard:Roche: Honoraria; Janssen: Honoraria. Cymbalista:Janssen: Honoraria, Research Funding; Gilead: Honoraria; Roche: Honoraria; Karyopharm: Honoraria. Letestu:Alexion: Honoraria, Research Funding; Roche: Honoraria.

Abstract 3052: NAMPT inhibition induces mitochondrial dysfunction leading to apoptosis in chronic lymphocytic leukemia cells

Abstract We have previously shown that the NAD depletion following inhibition of NAMPT by the NA-mimetic FK866 leads to loss of mitochondrial membrane potential, depletion of cellular ATP, cytochrome C release and caspase activation in primary Chronic Lymphocytic Leukemia (CLL) cells in vitro, and that these events are coupled with an increased production of reactive oxygen species uncharacteristic of metabolic inhibition. In the present study we characterize the effect of NAD depletion on CLL mitochondria and the downstream mechanism of cell death. We profiled the mitochondrial respiration of CLL cells and control B-lymphocytes, by extracellular flux analysis, over the 48 hours following FK866 treatment and found time-dependant inhibition of respiratory capacity consistent with disruption of the electron transport chain (ETC) in CLL cells. This led to suppression of basal respiration concomitant with the rise in ROS and approximately one day before loss of cellular viability. Interestingly, Zap70-positive CLL cells exhibited increased mitochondrial respiration and respiratory capacity over Zap70-negative CLL cells, but responded similarly to NAMPT inhibition. Additionally, mitochondrial respiration was not effected by NAMPT inhibition in control B-lymphocytes. Increase autophagic flux, caspase-dependant apoptosis and mitochondrio-nuclear translocation of Apoptosis Inducing Factor (AIF) were detected by western blot. However, neither pan-caspase inhibition with Z-VAD-fmk nor inhibition of autophagy with 3-methyladenine were sufficient to prevent FK866-induced CLL cell death. We conclude that NAD depletion following inhibition of NAMPT leads to disruption of the mitochondrial ETC in CLL cells, leading to mitochondrial initiation of the intrinsic apoptosis pathway. As healthy B-lymphocytes are resistant to this NAD depletion, these downstream effects are also selective for malignant cells. We are currently working to characterize the roles of NAD and Zap70 in the regulation and function of the CLL ETC. This will not only lead to an improved understanding of CLL metabolism, but will also inform new therapeutic strategies that will effectively employ NAMPT inhibition to treat this yet-incurable disease. Citation Format: Eric DJ Bouchard, Edgard M. Mejia, Iris Gehrke, Armando G. Poeppl, Donna Hewitt, James B. Johnston, Spencer B. Gibson, Grant M. Hatch, Versha Banerji. NAMPT inhibition induces mitochondrial dysfunction leading to apoptosis in chronic lymphocytic leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3052. doi:10.1158/1538-7445.AM2015-3052

Do Regulatory T Cells Have a Role in Disease Progression and Autoimmune Cytopenias of Chronic Lymphocytic Leukemia?,

Abstract 3864 Introduction:Chronic lymphocytic leukemia (CLL) originates from mature B lymphocytes which avoid death through the intercession of external signals, including T cells. There is improved understanding of the relationship between different cell types in the normal immune response and the dynamic cross talk between tumors and immune system regulating tumor growth. Cancer and autoimmunity are considered as anti-thesis of each other. Regulatory T cells (Tregs) are shown to have a protective role in autoimmune diseases and conversely known to promote oncogenesis. The exact role of Tregs in disease biology of CLL is still unclear. Aims and Objectives : to define the role of Tregs in clinical presentation, disease progression and autoimmune cytopenias in CLL. Materials and Methods : 32 treatment naive CLL patients and 10 normal healthy volunteers were evaluated in this prospective study. Investigations included clinical examination, biochemical tests, complete blood counts, bone marrow examinaton and multicolor flowcytometry (FCM). More stringently defined specific markers (CD4+CD25highCD127low FoxP3+) were used to enumerate Tregs. Routine CLL panel for diagnosis and prognosis included 17 monoclonal antibodies (MoAbs) labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) or peridinin chlorophyll protein (PerCP). FCM analysis was performed on FACS Calibur flowcytometer (BD Biosciences) using CellQuest® software. Diagnosis of autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia (ITP) and pure red cell aplasia (PRCA) was made according to standard criteria. Results : Study group included 20 male and 12 female CLL patients. The mean age was 64.2 years; with 8/32 (25%) patients being <55 years. 17/32 (53.12%) patients were categorized to Binet stage A and 20/32 (62.5%) were asymptomatic at diagnosis. Mean total leukocyte counts (TLC) and absolute lymphocyte counts (ALC) were 69,731/μl and 62,697/μl respectively. Mean hemoglobin and platelet counts were 12.1 g/dl and 178,087/μl respectively. 25 patients completed follow-up investigations at 6 months. Disease progression occurred in 12/25 (48%) patients, of which 5/25 (20%) patients progressed to Binet stage C and had symptoms other than lymphadenopathy while the number of asymptomatic patients decreased to 10/25 (40%). Disease progression was assessed according to NCI-WG guidelines. Autoimmune cytopenias occurred in 8/32(25%) patients. 3 of them at disease presentation and remaining 5 were diagnosed on follow-up. AIHA was diagnosed in one, ITP in 4 and PRCA in 3 patients. Mean Tregs % was lower in CLL patients (0.98+0.94) as compared to the controls (2.89+1.46) (p=0.0026). However, CLL patients had significantly higher absolute Treg cell count than the controls (581.63+734.5 vs. 83.89+55.45) (p=0.0006). While the mean % Treg increased by 46% (not significant, p >0.05) from stages A and B (early CLL) to stage C (advanced CLL), increase in mean ALC was 172% (p<0.005). The increase in mean absolute Treg count was further significantly higher 384% (p<0.005). There was no significant difference between the mean absolute Tregs count at baseline of CLL patients with disease progression (progressors) and without disease progression (nonprogressors) at follow-up. Absolute Treg count was significantly higher in patients having autoimmune cytopenias at presentation as well as on follow-up. Mean absolute Treg counts were not significantly different between ZAP70 positive and negative CLL patients (p=0.1), as well as between CD38 positive and negative CLL (p=0.15). There was a strong inverse correlation between lymphocyte doubling time and absolute Treg count (p=0.03) as well as lymphocyte doubling time and % Treg cells (p=0.006). No correlation could be found between absolute Treg count and CD4: CD8 ratio, r= 0.29 (p=0.1). Conclusion : Absolute Treg cell counts (rather than % Treg) are increased in CLL patients compared to controls and also increased with disease severity. Absolute Treg counts may be more important than % Tregs in CLL pathogenesis. Tregs also have a strong inverse relation with lymphocyte doubling time, also indirectly confirming the role of Tregs in disease progression. We also found Tregs to be increased in patients with autoimmune cytopenias. Autoimmune cytopenias may be viewed as a continuum of CLL disease itself rather than a complication of advanced disease. Disclosures:No relevant conflicts of interest to declare.

Relative Prognostic Significance of ZAP-70 and IGHV1-69 Expression in Chronic Lymphocytic Leukemia,

Abstract 3891The leukemia B cells of patients (pts) with chronic lymphocytic leukemia (CLL) express a restricted repertoire of immunoglobulin heavy chain variable region (IGHV) genes. Moreover, certain IGHV genes appear over-represented in this repertoire. Among these, IGHV1-69 was first identified as most frequently expressed, being used by the CLL cells of nearly 20% of all pts (PNAS, 86:5913–7, 1989). CLL cells most frequently express IGHV1-69 without somatic mutations and often with restricted D and JH segment use, providing for stereotypic motifs in the heavy chain third complementarity determining region (HCDR3). We addressed whether there were peculiar biologic or clinical features of pts with CLL that used IGHV1-69. For this, we studied 452 pts identified in a cohort of 2,866 followed by the CLL Research Consortium (CRC) found to have CLL cells that express IGHV1-69. This accounted for 16% of all pts. We found that 420 of 452 CLL samples (93%) express IGHV1-69 without somatic mutation (≥98% sequence homology with germline IGHV1-69), which is in significant contrast to the frequency use of UM IGHV among CLL samples that do not use IGHV1-69. As noted for CLL pts in general, there is a strong association between mutation status and clinical behavior. Among pts that use IGHV1-69, the 32 of 452 pts that use MU IGHV1-69 had a highly indolent clinical course, with a median time from diagnosis to initial treatment (TFS) of 17.3 years. This was significantly longer than the median TFS of 2.8 yrs for the 452 pts that used UM IGHV1-69 (p<0.0001). Among the pts that used UM IGHV1-69 we identified a stereotypic HDCR3 motif shared by more than one patient in 249/452 (55%) of the cases. Multivariate analysis failed to discriminate any significant differences in the median TFS of pts that used IGHV1-69 that had a stereotypic CDR3 motif versus pts who had an idiosyncratic HCDR3 (2.9 yrs vs 3.0 yrs, respectively p=0.14). Interphase FISH for common cytogenetic aberrations in CLL were available for 281 of the 452 cases. Among these, 58% of the cases had deletions at 17p (18%), 11q (23%), or trisomy 12 (17%). The remaining cases had no detectable chromosomal abnormalities (24%) or isolated deletion of 13q (18%).The CLL cells of all 452 pts were examined for ZAP-70. There was a strong association between the expression of ZAP-70 and use of UM IGHV1-69. However, the association between ZAP-70 expression and use of UM IGHV1-69 was not absolute. Only 70% with UM IGHV1-69 were ZAP-70 positive, as were 12.5% that used M IGHV1-69. Of all 452 pts that expressed IGHV1-69, 300 (66%) had ZAP-70 positive CLL cells. These pts had a median TFS of 2.3 yrs, which was significantly shorter than that of the remaining 152 pts with ZAP-70-negative CLL, who had a median TFS of 4.3 yrs (p<0.0001). Moreover, of the 420 pts that used UM IGHV1-69, 296 (70%) had CLL cells that expressed ZAP-70; these pts had a median TFS of 2.3 yrs. This was significantly shorter than the median TFS of pts with CLL cells that express UM IGHV1-69, but were ZAP-70 negative 4.1 yrs (p<0.0001).A Cox regression model revealed that although the presence of detectable chromosomal aberrations was associated to a shorter median TFS, ZAP-70 was a stronger predictor of short TFS (HR for 13q =1.1 p =0.03, HR for trisomy 12 =1.2 p =0.03, HR for 11q =1.6 p =0.03, HR for 17p =1.8, p =0.03) (HR for ZAP-70 positive = 1.8, p=0.0004).The Cox regression model was used to assess the associations of ZAP-70, and the use or not of the UM IGHV1-69 gene with TFS (p-value <0.05 were considered as significant). We investigated these associations using a previously published cohort of characterized 705 CLL pts (Blood.2008;112:1923). The HR associated with the expression of ZAP-70 (HR=3.2) (p<0.0001) was significantly higher than if either the UM IGHV1-69 or the cases with UM IGHV other than IGHV1-69 were incorporated into the model (HR=1.9 and HR=1.6 respectively, p=0.001).We conclude that cases that use IGHV1-69 are peculiar in that they more frequently use UM IGHV and appear to have a higher frequency of adverse cytogenetic features than CLL cases at large. In addition, we found that CLL-cell expression of ZAP-70 can segregate pts that use UM IGHV1-69 into subgroups with disparate clinical behavior, despite the fact that all patients use the same IGHV gene. Moreover, multivariable analyses revealed that ZAP-70 was strongest predictor of short TFS among all other considered prognostic parameters in this distinctive cohort of pts. Disclosures:Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

ZAP-70 enhances migration of malignant B lymphocytes toward CCL21 by inducing CCR7 expression via IgM-ERK1/2 activation

AbstractZAP-70 in chronic lymphocytic leukemia (CLL) has been associated with enhanced B-cell receptor (BCR) signaling, survival, and migration. We investigated whether ZAP-70 can directly govern migration and the underlying mechanisms. In the ZAP-70 stably transfected Ramos cell line, IgM stimulation, but no IgD, enhanced phosphorylation of ERK1/2, Akt and Syk, and delayed IgM and CD79b internalization. In contrast, in the Raji cell line, where ZAP-70 was constitutively phosphorylated, ERK1/2, but not Akt, was phosphorylated, suggesting that MAPK pathway mediates ZAP-70 effects. BCR stimulation modulated the expression of CCR7, CXCR4, CXCR5, CD44, CD49d, and CD62L, which were up-regulated in ZAP-70–positive CLL primary subclones. The most dramatic change after BCR engagement in ZAP-70-transfected cells was CCR7 up-regulation, this being impaired by ERK1/2 inhibition and translating into both increased signaling and migration toward CCL21. Primary CLL subclones with high ZAP-70 expression showed increased migration toward CCL21. In conclusion, ZAP-70 ectopic expression led to enhanced BCR signaling after IgM stimulation and increased the expression of CCR7 predominantly via ERK1/2, increasing the response and migration toward CCL21. In primary CLL samples, cellular subsets with high ZAP-70 expression had increased expression of adhesion molecules and chemokine receptors in addition to an enhanced ability to migrate toward CCL21.

The expression of SOX1 1,cyclin D1, cyclin D2 and cyclin D3 in B-cell lymphocytic proliferative diseases

：Objective To investigatethe expression levels of the SOX11 and cyclin D isoforms in Bcell lymphocyticproliferative disease (B-LPD). Methods We assessed the expressions of SOX11, cyclins D1,cyclin D2, cyclin D3 mRNA using real-time quantitative reverse transcriptase-polymerasechain reaction (qRT-PCR) and SOX11 protein using immunohistochemistry. Results Data showedthe transcriptional level of SOX11 was higher for the mantle cell lymphoma (MCL) samplescompared with the groups of chronic lymphocytic leukemia (CLL), diffuse large B celllymphoma (DLBCL), hairy cell leukemia (HCL), splenic marginal zone lymphoma (SMZL) andhealthy collators (P1 =0. 002, P2 =0. 017, P3 =0. 015, P4 =0. 008,P5 = 0. 008,respectively). Also MCL showed higher transcript level of cyclin D1 than CLL, DLBCL,HCL,SMZL and healthy collator group (P1 ＜0. 001, P2 = 0.008, P3 = 0. 020, P4 ＜ 0. 001, P5 = 0.005,respectively). The cyclin D1 and cyclin D2 expression levels were both higher in DLBCLgroup than in SMZL group (P1 =0. 049, P2 =0. 005, respectively). The SOX11 expressionlevel was highly related with the expression levels of cyclin D1, cyclin D2 and cyclin D3(r1 = 0. 444, P1 ＜0. 001 ; r2 = 0. 279, P2 ＜0.001; r3 =0. 425, P3＜0. 001, respectively). Moreover, 3 out of 4 MCL patients showed nuclear stainingforSOX11, while other 11 B-LPD examples were negative, including 7 cases of DLBCL, 2 casesof mucosaassociated lymphoid tissue (MALT) lymphoma, 2 cases of SMZL. Futhermore, we alsofound the ZAP70-positive CLL patients had higher SOX11 expression levels than thoseZAP70-negative CLL patients (P1 =0. 021). Conclusion It was revealed that MCL patients hada higher expression levels of SOX11 and cyclin D1 mRNA, and specially expressed nuclearSOX11 protein. Key words: lymphocytic proliferative disease; mRNA ; SOX11; cyclinD1; cyclin D2; cyclin

ZAP‐70 expression is associated with increased risk of autoimmune cytopenias in CLL patients

Autoimmune cytopenias (AIC) are frequent in chronic lymphocytic leukemia (CLL) patients, but risk factors and prognostic relevance of these events are controversial. Data about the influence on AIC of biological prognostic markers, as ZAP-70, are scanty. We retrospectively evaluated AIC in 290 CLL patients tested for ZAP-70 expression by immunohistochemistry on bone marrow biopsy at presentation. They were 185 men, median age 63 years, 77.9% Binet stage A, 17.6% B and 4.5% C. AIC occurred in 46 patients (16%): 31 autoimmune hemolytic anemias, 10 autoimmune thrombocytopenias, four Evans syndromes, and one pure red cell aplasia. Of the 46 cases of AIC, 37 (80%) occurred in ZAP-70 positive patients and nine (20%) in ZAP-70 negatives. ZAP-70 expression [Hazard Ratio (HR) = 7.42; 95% confidence interval (CI): 2.49-22.05] and age >65 years (HR = 5.41; 95% CI: 1.67-17.49) resulted independent risk factors for AIC. Among the 136 patients evaluated both for ZAP-70 expression and IGHV status, the occurrence of AIC was higher in ZAP-70 positive/IGHV unmutated cases (35%) than in patients ZAP-70 negative/IGHV mutated (6%) or discordant for the two parameters (4%; P < 0.0001). In ZAP-70 positive patients, occurrence of AIC negatively influenced survival (HR = 1.75; 95% CI: 1.06-2.86). The high risk of developing AIC in ZAP-70 positive CLL, particularly when IGHV unmutated, should be considered in the clinical management.

Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells.

BackgroundZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method).MethodsCytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set).ResultsThe optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70+ cases, respectively. Univariate analyses resulted in a better separation of ZAP-70+ vs. ZAP-70- CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70+ cases, respectively. ZAP-70+ patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70- CLL, and to cases classified ZAP-70+ by the T-method only.ConclusionsWe suggest to evaluate ZAP-70 expression in routine settings using the T/B Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B Ratio value as optimal cut-off to discriminate ZAP-70+ (T/B Ratio less than 3.0) from ZAP-70- (T/B Ratio more/equal than 3.0) cases.

Steroid effects on ZAP-70 and SYK in relation to apoptosis in poor prognosis chronic lymphocytic leukemia

There is resurgent interest in glucocorticoids (GCs) in the treatment of poor prognosis chronic lymphocytic leukemia (CLL). Little is known however on how GCs induce apoptosis in CLL. Methylprednisolone (MP) induces apoptosis in ZAP-70 positive CLL more readily than in ZAP-70 negative CLL, which is in contrast to the effects of radiation and chemotherapy. The increased GC sensitivity of ZAP-70 + CLL was studied in relation to the expression status of ZAP-70 and the related signal transducing tyrosine kinase SYK. Both ZAP-70 and SYK were downregulated by GC treatment. Moreover, SYK was dephosphorylated by the phosphatase PTP1B of which the expression and translation levels were induced by GCs. Inhibition of PTP1B successfully restored ZAP-70 expression and SYK phosphorylation but did not interfere with GC-induced apoptosis. Therefore, the downregulation of ZAP-70 and P-SYK per se during treatment with GCs is not sufficient to induce apoptosis, and different mechanisms must therefore be responsible for the increased steroid sensitivity of ZAP-70 + CLL.

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.

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

AbstractLeukemia-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.

Consolidation and Maintenance Immunotherapy with Rituximab Improve Progression Free Survival within ZAP-70 Positive Chronic Lymphocytic Leukemia (CLL).

Clinical studies of monoclonal antibodies combined with chemotherapy have attained more complete responses and longer duration of responses in CLL because this approach reduces disease burden to levels detectable only by immunological or molecular methods. Recent literature data indicate that VH genes, CD38, ZAP-70 protein, and cytogenetic status have a major impact on CLL prognosis. We performed a phase II study that added rituximab sequentially to fludarabine (Flu) as initial therapy for symptomatic, untreated CLL in order to evaluate both the clinical response and outcome. Remission status was also assessed by a multiparametric flow cytometric method based on the detection of CD19+CD5+CD79b− residual B-CLL lymphocytes. VH mutational status, CD38 and cytogenetics were obtained in all pts before treatment. ZAP-70 protein was performed by flow cytometry using an anti-ZAP-70 Alexa Fluor 488 conjugated antibody. Seventy-five CLL pts, median age 60 years, received six monthly courses of Flu (25 mg/m2 for 5 days) and four weekly doses of rituximab (375 mg/m2) starting on an average of thirty days after completion of Flu therapy. According to modified Rai stages, 6 pts had a low stage, 66 an intermediate stage and 3 a high stage. Based on NCI criteria, 61/75 (81%) pts achieved a complete remission (CR), 10/75 (13%) a partial remission (PR) and 4/75 (5%) no response. Three pts presented grade 3 (WHO) infective lung toxicity and 1 patient acute fatal B hepatitis.Hematologic toxicity included neutropenia (grade 3 and/or 4 in 38 pts) and thrombocytopenia (grade 3 and/or 4 in 4 pts). Twenty eight pts, either with CD5+CD19+CD79b− (MRD) bone marrow (BM) cells >1% (n=17 pts) or with CD19+CD5+CD79b− (MRD) peripheral blood lymphocytes (PBL) >1000/microl (n=11 pts) within six months after completion of the induction treatment, underwent consolidation/maintenance therapy with four monthly cycles of rituximab at 375 mg/m2 followed by twelve monthly doses of rituximab at 150 mg/m2. The median follow-up duration was 37 months. Noteworthy, all pts experienced a long progression-free survival (PFS) from treatment (67% at 5 years). Nevertheless, CLL pts that underwent consolidation therapy (n=28) showed a significant longer duration of response (75% vs 27% at 5 years, P=0.002) in comparison with the subset of not consolidated and BM or PBL MRD positive (n=18) CLL pts. Interestingly, BM and PBL MRD negative pts (n=26) showed a duration of response similar to that of the consolidated pts. Moreover, a significant shorter PFS was observed within CD38+ pts (29% vs 79% at 5 years, P=0.005), unmutated pts (0% vs 92% at 2 years, P=0.001), ZAP-70+ pts (29% vs 92% at 5 years; P=0.00003) and within the “poor risk” [trisomy 12 or del 11q or del 17p] cytogenetic subset (0% vs 79% at 2 years, P=0.03). Interestingly, within the ZAP-70+ subset (n=35), the consolidated pts (n=12) showed a longer duration of response (68% vs 0% at 2.6 years, P=0.007) in comparison with the unconsolidated pts (n=11). Therefore, the addition of a consolidation/maintenance therapy with rituximab prolongs significantly the duration of response allowing a better outcome. Moreover, rituximab improves significantly PFS of pts notoriously at worse prognosis, such as ZAP-70+ B-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.

ZAP-70 Expression in Chronic Lymphocytic Leukemia (CLL) Is Independent of Rai Stage: A Retrospective Review of 38 Patients (pts).

ZAP-70 is a tyrosine kinase protein involved in T cell signaling, overexpression of which is associated with a poor prognosis in CLL. Clinical information on 32 pts with CLL, and 6 pts with prolymphocytic transformation (PLT) followed at our institution was retrospectively reviewed for Rai stage, ZAP-70, CD38, FISH and response to treatment to determine the prognostic association among these factors. Ages ranged from 37 to 78 (median 59). 5/32 pts with CLL had received prior initial therapy, while 17/19 pts had not. They eventually were all treated with fludarabine based regimens either as salvage or upfront. The remaining 8/32 had not required therapy. Response to therapy was correlated with ZAP-70 expression as measured by immunohistochemistry using a monoclonal antibody to ZAP-70 (clone 2F3.2).17/32 patients (53%) were ZAP-70 positive, as were 3/6 (50%) of the patients with PLT. Of the patients with CLL who were ZAP-70 positive, 5 (30%) were stage 0-II, and 12 (70%) were stage III/IV, while18% did not require therapy. Of the 13 who were treated there were 4 (31%) CR, and 5 (38%) PR. Of the patients who were ZAP-70 negative, 50% (7/14) had stages 0-II and 50% had stage III/IV disease. 5/14(36%) were not treated. Of the 9 who were treated there were 2 CR 22%, and 7 PR 78% (p=0.01). 3 PLT patients were previously treated for CLL with fludarabine-based combinations. When they required therapy for PLT, two achieved a CR (one to rituximab/fludarabine/cytoxan_ZAP-70 positive patient, and one to R-CHOP combination_ZAP-70 negative), and another a PR (to R-EPOCH combination_ZAP-70 negative patient). Of the previously untreated PLT patients, two ZAP-70 positive patients achieved a CR (one to rituximab/fludarabine/cyclophosphamide, and one to R-CHOP). One ZAP-70 negative patient was refractory to fludarabine/rituximab combination.Conclusion: ZAP-70 positive CLL is more likely to be refractory to treatment, while ZAP-70 negative pts achieved a higher ORR (p=0.01). Although pts who were ZAP-70 positive were more likely to have advanced stage, 30% of these pts had early stage disease. As such, ZAP-70 did not closely correlate with stage, and may be an independent prognostic factor in these pts. Response to treatment could also not be predicted by ZAP-70 expression.ZAP-70Number of PtsPrevious TreatmentNumber of PtsResponsePositive16No103CR, 4PRYes31CR, 1PRNegative14No72CR, 5PRYes22PR

Clinical Characteristics and Outcome of a Large Series of Patients with Chronic Lymphocytic Leukemia (CLL) According to ZAP-70 Expression.

The prognosis of patients with CLL has been traditionally assessed by using clinical stages. Clinical stages, however, are a mere reflection of the biological diversity of CLL. In this regard, we and others have recently shown that ZAP-70 expression separates CLL into two clinical forms with different outcome. The aim of this study was to analyze the distribution of age, gender, and different prognostic parameters according to ZAP-70 expression in 222 pts with CLL (median age, 62 yrs; female/male: 89/133). ZAP-70 was determined by flow cytometry, a high expression being defined as >20% ZAP-70 positive CLL cells. Median follow-up of the series was 7.1 years. Clinical stages at diagnosis were: Binet stage A, 187 pts; B, 21 pts; and C, 14 pts. Rai Stage 0, 123 pts; I, 53 pts; II, 28 pts; III, 7 pts; and IV, 11 pts. Overall median survival was 13 years. Parameters associated with a high ZAP-70 expression were lymphocyte doubling time >12 mos., atypical morphology, bone marrow infiltration > 60%, increased serum LDH and timidin kinase, CD38 expression >20%, del(13q14), unmutated status of IgVH, and development of Richer syndrome. Interestingly, ZAP-70 expression distribution correlated with Rai's stage (0 vs. 1 to 4). Also, CD38 expression was able to separate two subpopulations with different survival among CLL cases with low ZAP-70 expression (median survival 18 yrs vs. 13 yrs), but not in pts. with ZAP-70>20%. About 50% of pts in Binet A progressed during the follow-up. Median time to progression for patients with high ZAP-70 expression was 3.3 years whereas it was not reached for patients with low ZAP-70 expression. Median survival of pts with low ZAP-70 expression was 16.3 years, whereas it was of 8.5 years in those with high ZAP-70 expression. In conclusion, ZAP-70 expression identifies a subgroup of patients with CLL with unfavorable prognostic factors related to the proliferation and progression of the disease.Prognostic Parameters according to ZAP-70 expressionVariableZAP-70 >20%ZAP-70 < 20%p=Median age60 yrs63 yrsGender (female)39%41%LDT < 12 mos42%23%0.009Atypical morphology54%12%0.000BM infiltration >60%50%33%0.03Increased LDH13%4%0.028Increased TK66%28%0.000CD38 > 20%67%32%0.000Del(13q14)38%55%0.027Unmutated IgVH88%11%0.000Richter Syndrome8%2%0.039Rai Stage > 053%37%0.039TTP (Binet A)3.3 yrsNR0.000Median Survival16.3 yrs8.5 yrs0.000