No convincing evidence for a role of CD31-CD38 interactions in the pathogenesis of chronic lymphocytic leukemia

Although CD38, a marker of poor prognosis in chronic lymphocytic leukemia (CLL), is known primarily as an ecto-enzyme, it has also been ascribed a receptor function. Interaction with its proposed ligand CD31 expressed on nurse-like cells would result in proliferative and survival-signals. Yet, in CLL, both homotypic and heterotypic CD31-CD38 interactions are expected to be rather ubiquitous. We analyzed whether CD38-CD31 interactions result in proliferative and antiapoptotic signals. We found a high expression of CD31 on CLL, irrespective of CD38 expression. Coculture of CD38^high CLL with endothelial cells or CD31 transfected fibroblasts, with or without blocking CD31 or CD38 antibodies, did not result in increased survival or proliferation. Analysis of gene expression of most known regulators of apoptosis revealed no influence of coculture with CD31-expressing feeder cells. In conclusion, our data do not support an important contribution of CD38 triggering by CD31 to the proliferative and antiapoptotic state of the leukemic clone.     

Increased CD39 expression on CD4+ T lymphocytes has clinical and prognostic significance in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes proinflammatory extracellular ATP, generates anti-inflammatory adenosine, and also protects regulatory T cells from ATP-induced cell death. In this study, we investigated the clinical significance of CD39 expression on CD4(+) T cells in 62 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4(+)CD39(+) lymphocytes were increased in the peripheral blood of patients with CLL and correlated with the advanced stage of disease. CD4(+)CD39(+) cells were also higher in patients with CLL, who needed therapeutic intervention, and in those who had unmutated immunoglobulin heavy chain variable region gene, were ZAP70(+) or had β2-microglobulin levels of >3 g/L. There were more CD4(+)CD39(+) lymphocytes in the bone marrow compartment than in the peripheral blood, and in vitro studies showed that CD39 can be induced on CD4(+) cells by exposure to ATP or indirectly, following B cell receptor engagement. This may support the notion that the leukemic cells contribute to create an immune-subversive environment, and perhaps to a poorer prognosis. CD39(+) may also serve as a future target for the development of novel therapies with immune-modulating antitumor agents in CLL.     

Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1

Chronic lymphocytic leukemia (CLL) B cells have defects in apoptosis pathways and therefore accumulate in vivo. However, when removed from the patient and cultured in vitro, these malignant cells rapidly undergo apoptosis. Recent studies suggest that leukemia cell survival is influenced by interactions with nonleukemia cells in the microenvironment of lymph nodes, marrow, and other tissues. To model such cell-cell interactions in vitro, we cultured freshly isolated CLL B cells with a follicular dendritic cell line, HK. CLL B cells cocultured with HK cells were protected from apoptosis, either spontaneous or induced by treatment with anticancer drugs. Protection against spontaneous apoptosis could also be induced by coculturing the CLL B cells with normal dendritic cells (DCs) or with a CD40-ligand (CD154)-expressing fibroblast cell line. Examination of the expression of several apoptosis-regulatory proteins revealed that coculture with HK cells or DCs induced up-regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in CLL B cells, whereas CD40 ligation increased expression of Bcl-X(L). Cell-cell contact was required for HK-induced protection, and introducing neutralizing antibodies against various adhesion molecules showed that CD44 was involved in HK-mediated survival, whereas CD40, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were not. Anti-CD44 antibodies also blocked Mcl-1 induction by HK cells. Mcl-1 antisense oligonucleotides reduced leukemia cell expression of Mcl-1, and significantly suppressed HK-induced protection against apoptosis, whereas control oligonucleotides had no effect. Thus, HK cells protect CLL B cells against apoptosis, at least in part through a CD44-dependent mechanism involving up-regulation of Mcl-1, and this mechanism is distinct from that achieved by CD40 ligation. Consequently, the particular antiapoptotic proteins important for CLL survival may vary depending on the microenvironment.     

Expression of CD27 and its ligand, CD70, on chronic lymphocytic leukemia B cells

Crosslinking the CD27 antigen on T cells provides a costimulatory signal that, in concert with T-cell receptor crosslinking, can induce T- cell proliferation and cellular immune activation. We find that chronic lymphocytic leukemia (CLL) B cells from most patients coexpress both membrane-bound and soluble CD27, along with its newly identified ligand, CD70. The expression of soluble CD27 may preclude leukemic B cells from stimulating T cells via CD70, thereby potentially impairing their ability to function as effective antigen-presenting cells. We find that leukemic B-cell expression of soluble and membrane-bound CD27 can be downmodulated through a CD40-dependent signal. This signal also induces enhanced expression of CD70 on both normal and leukemic B cells. We find that tumor necrosis factor (TNF)-alpha, or the Th1 cytokine interferon (IFN)-gamma, also can induce downmodulation of CD27, whereas Th2-associated cytokines interleukin-4 (IL-4) or IL-10 can enhance leukemic B-cell expression of this accessory molecule. The modulation of CD27 induced by these conditions is accompanied by reciprocal changes in the expression levels of CD70, suggesting that these accessory molecules may be engaged in reciprocal receptor-ligand downmodulation. Consistent with this, we observe that co-culture of CLL B cells with transfected murine plasmacytoma cells that express human CD70 affects downmodulation of CD27 and enhanced expression of CD70 on leukemic B cells, but does not affect expression of CD27 mRNA. However, we find that CD40-crosslinking, in addition to reducing the level of CD27 protein, also reduces leukemic B-cell expression of CD27 mRNA. This argues that the changes in the expression levels of CD27 following CD40-signaling are not simply due to induced increases in the expression levels of CD70. Finally, we demonstrate that reciprocal changes in expression of CD27 and CD70 may contribute to the enhanced antigen-presenting capacity of CLL B cells after CD40-dependent leukemic B-cell activation. These findings expand the understanding of the regulation of costimulatory molecules important in antigen presentation and also have implications for the immunobiology of and therapy for CLL.     

A proliferation-inducing ligand (APRIL) serum levels predict time to first treatment in patients affected by B-cell chronic lymphocytic leukemia

Purpose: A proliferation‐inducing ligand (APRIL), a tumor necrosis factor superfamily member involved in B‐lymphocytes differentiation and survival, plays a role in protecting B‐Cell Chronic lymphocytic leukemia (B‐CLL) cells from apoptosis. Having observed that APRIL serum (sAPRIL) levels were higher in B‐CLL patients with CLL at diagnosis as compared to healthy donors (14.61 ± 32.65 vs. 4.19 ± 3.42 ng/mL; P < 0.001), we tested the correlation existing in these patients between sAPRIL, clinical–biological parameters and disease progression. Experimental design: sAPRIL levels were measured by ELISA in 130 patients with B‐CLL at diagnosis and in 25 healthy donors. Results: sAPRIL levels did not correlate with gender, age, clinical stage, blood cell counts, β2‐microglobulin (β2M) levels, ZAP‐70 and CD38 expression. Using median sAPRIL natural logarithm (ln) as cutoff, we distinguished two groups of patients (APRIL_LOW and APRIL_HIGH) who were comparable with regard to clinical–biological parameters and overall survival, but different with regard to time to the first treatment (TTFT; P = 0.035). According to univariate analysis, high lymphocyte count, high β2M, Binet stage B–C, ZAP‐70 expression and ln(sAPRIL) above median were associated with earlier TTFT. Advanced clinical stage, high β2M, ZAP‐70 expression and ln(sAPRIL) above median remained independently predictive of shorter TTFT at multivariate analysis. Moreover, sAPRIL increased its prognostic significance when patients were stratified according to independent favorable clinical–biological characteristics (low β2M, stage A and lack of ZAP‐70 expression). Conclusions: sAPRIL is a novel indicator of shorter TTFT in B‐CLL and a predictor of progression especially in patients otherwise considered at low risk according to validated prognostic factors.     

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

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

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

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

Igbeta(CD79b) mRNA expression in chronic lymphocytic leukaemia cells correlates with immunoglobulin heavy chain gene mutational status but does not serve as an independent predictor of clinical severity

The etiology of chronic lymphocytic leukemia (CLL) is poorly understood and its course is highly variable. Somatic hypermutation (SHM) of the immunoglobulin heavy chain (IgV(H)) gene and ZAP70 protein expression have been reported as prognostic indicators. However, these assays are not widely available and their concordance is imperfect. Thus a need exists to identify additional molecular determinants of CLL. The Igbeta (CD79b) subunit of the B cell antigen receptor is essential for B lymphocyte function. Defects in Igbeta expression are implicated in CLL pathogenesis. We have analyzed Igbeta mRNA expression in CLL cells in 40 consecutive patient samples. About 75% of the samples showed the expected decrease of Igbeta surface staining. Igbeta mRNA levels covered a wider range, did not correlate with Igbeta surface staining, but clearly distinguished the normal and CLL lymphocyte populations. Remarkably, Igbeta mRNA levels correlated strongly with SHM; Igbeta mRNA levels in CLL cells were significantly higher in patients with an unmutated IgV(H) gene when compared with those in whom IgV(H) was hypermutated (P = 0.008). In contrast, no correlation was observed between Igbeta mRNA levels and ZAP70 expression. Multiple parameters abstracted from chart reviews were used to estimate severity of CLL in each case. While severity correlated strongly with ZAP70 staining, and to a lesser extent with SHM status, there was no correlation with Igbeta mRNA levels. These data establish a strong linkage between Igbeta mRNA expression and SHM in CLL and highlight the complex relationships between biochemical parameters and clinical status in this disease.     

Circulating microenvironment of CLL: Are nurse-like cells related to tumor-associated macrophages?

B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common hematologic malignancies in Western countries. Accumulation of leukemic lymphocytes in peripheral blood, bone marrow and secondary lymphatic organs of CLL patients is due to decreased apoptosis rather than to increased proliferation. The former is driven by signals from a specific microenvironment, created by stromal cells of mesenchymal origin, follicular dendritic cells, T lymphocytes and others.Nurse-like cells (NLCs) were first described to differentiate from peripheral blood mononuclear cells of CLL patients in vitro, then they have been also found in proliferation centers of their lymphatic tissues. Like tumor-associated macrophages (TAMs) in solid tumors, nurse-like cells promote survival of CLL lymphocytes. NLC gene expression patterns suggest their similarity to TAMs and differ between patients depending on ZAP70 protein expression status. NLC number in vitro corresponds with CD14 expressing cell count and beta-2-microglobulin serum level, and positively correlates with leukemic lymphocyte viability. As NLCs strongly express genes for adhesion molecules and secrete chemokines of antiapoptotic activity, they should be considered as a target for anti-microenvironment therapy of this incurable disease.     

Defining the prognosis of early stage chronic lymphocytic leukaemia patients

Approximately 70% of chronic lymphocytic leukaemia (CLL) patients present with early stage disease, therefore defining which patients will progress and require treatment is a major clinical challenge. Here, we present the largest study of prognostic markers ever carried out in Binet stage A patients (n = 1154) with a median follow‐up of 8 years. We assessed the prognostic impact of lymphocyte doubling time (LDT), immunoglobulin gene (IGHV) mutation status, CD38 expression, ZAP‐70 expression and fluorescence in situ hybridization (FISH) cytogenetics with regards to time to first treatment (TTFT) and overall survival (OS). Univariate analysis revealed LDT as the most prognostic parameter for TTFT, with IGHV mutation status most prognostic for OS. CD38 expression, ZAP‐70 expression and FISH were also prognostic variables; combinations of these markers increased prognostic power in concordant cases. Multivariate analysis revealed that only LDT, IGHV mutation status, CD38 and age at diagnosis were independent prognostic variables for TTFT and OS. Therefore, IGHV mutation status and CD38 expression have independent prognostic value in early stage CLL and should be performed as part of the routine diagnostic workup. ZAP‐70 expression and FISH were not independent prognostic markers in early stage disease and can be omitted at diagnosis but FISH analysis should be undertaken at disease progression to direct treatment strategy.     

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. Am. J. Hematol. 2010. © 2010 Wiley‐Liss, Inc.     

Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765

B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in knockout mouse models its mutation has a relatively B cell-specific phenotype. Herein, we demonstrate that BTK protein and mRNA are significantly over expressed in CLL compared with normal B cells. Although BTK is not always constitutively active in CLL cells, BCR or CD40 signaling is accompanied by effective activation of this pathway. Using the irreversible BTK inhibitor PCI-32765, we demonstrate modest apoptosis in CLL cells that is greater than that observed in normal B cells. No influence of PCI-32765 on T-cell survival is observed. Treatment of CD40 or BCR activated CLL cells with PCI-32765 results in inhibition of BTK tyrosine phosphorylation and also effectively abrogates downstream survival pathways activated by this kinase including ERK1/2, PI3K, and NF-κB. In addition, PCI-32765 inhibits activation-induced proliferation of CLL cells in vitro, and effectively blocks survival signals provided externally to CLL cells from the microenvironment including soluble factors (CD40L, BAFF, IL-6, IL-4, and TNF-α), fibronectin engagement, and stromal cell contact. Based on these collective data, future efforts targeting BTK with the irreversible inhibitor PCI-32765 in clinical trials of CLL patients is warranted.     

MFI ratio estimation of ZAP-70 in B-CLL by flow cytometry can be improved by considering the isotype-matched antibody signal

Introduction: The IgV_H mutational status of B‐cell chronic lymphocytic leukemia (B‐CLL) is of prognostic value. Expression of ZAP‐70 in B‐CLL is a surrogate marker for IgV_H unmutated (UM). As determination of IgV_H mutational status involves a methodology currently unavailable for most clinical laboratories, it is important to have available a reliable technique for ZAP‐70 estimation in B‐CLL. Flow cytometry (FC) is a convenient technique for this purpose. However, there is still no adequate way for data analysis, which would prevent the assignment of false positive or negative expression. Methods: We have modified the currently most accepted technique, which uses the ratio of the mean fluorescent index (MFI) of B‐CLL to T cells. The MFI for parallel antibody isotype staining is subtracted from the ZAP‐70 MFI of both B‐CLL and T cells. We validated this technique comparing the results obtained for ZAP‐70 expression by FC with those obtained with quantitative PCR for the same patients. Results: We applied the technique in a series of 53 patients. With this modification, a better correlation between ZAP‐70 expression and IgV_H UM was obtained. Conclusions: Thus, the MFI ratio B‐CLL/T cell corrected by isotype is a reliable analysis technique to estimate ZAP‐70 expression in B‐CLL.     

Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression

It was hypothesized that contact between chronic lymphocytic leukaemia (CLL) B-cells and marrow stromal cells impact both cell types. To test this hypothesis, we utilized a long-term primary culture system from bone biopsies that reliably generates a mesenchymal stem cell (MSC). Co-culture of MSC with CLL B-cells protected the latter from both spontaneous apoptosis and drug-induced apoptosis. The CD38 expression in previously CD38 positive CLL B-cells was up-regulated with MSC co-culture. Upregulation of CD71, CD25, CD69 and CD70 in CLL B-cells was found in the co-culture. CD71 upregulation was more significantly associated with high-risk CLL, implicating CD71 regulation in the microenvironment predicting disease progression. In MSC, rapid ERK and AKT phosphorylation (within 30 min) were detected when CLL B-cells and MSC were separated by transwell; indicating that activation of MSC was mediated by soluble factors. These findings support a bi-directional activation between bone marrow stromal cells and CLL B-cells.     

In-tandem insight from basic science combined with clinical research: CD38 as both marker and key component of the pathogenetic network underlying chronic lymphocytic leukemia

The absence of mutations in the IgV genes, together with the presence of ZAP-70 and CD38, are the most reliable negative prognostic markers for chronic lymphocytic leukemia (CLL) patients. Several lines of evidence indicate that CD38 may be not only a diagnostic marker but also a key element in the pathogenetic network in CLL. First, CD38 is a receptor that induces proliferation and increases survival of CLL cells. Second, CD38 signals start upon interaction with the CD31 ligand expressed by stromal and nurse-like cells. Third, CD38/CD31 contacts up-regulate CD100, a semaphorin involved in sustaining CLL growth. Fourth, evidence that nurselike cells express high levels of CD31 and plexin-B1, the high-affinity ligand for CD100, offers indirect confirmation for this model of receptor cross-talk. Elements of variation in the clinical course of CD38(+) CLL patients include (1) potential intersection with ZAP-70, a kinase involved in the CD38 signaling pathway in T and natural killer (NK) cells, and (2) the effects of genetic polymorphisms of the receptors involved, at least of CD38 and CD31. Consequently, CD38 together with ZAP-70 appear to be the key elements of a coreceptor pathway that may sustain the signals mediated by the B-cell receptor and potentially by chemokines and their receptors. This would result in acquisition of increased survival potential, providing clues to the poorer prognosis of CD38(+) patients.     

Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells

Bacterial DNA and synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the immune system in a sequence-dependent manner. Here we investigated the potential of CpG-ODNs to cause proliferation, cytokine production, and regulation of surface molecules in human B-chronic lymphocytic leukemia (CLL) cells. CpG-ODN induced proliferation in both B-CLL cells and normal B cells; however, only B-CLL cells increased proliferative responses when CpG-ODN was added to co-cultures of CD40-ligand transfected mouse fibroblasts (CD40LF) and B cells. Production of interleukin-6 and tumor necrosis factor alpha was detectable at borderline levels, using CpG-ODN as the only stimulus. In contrast, when CpG-ODN was added to co-cultures of B cells and CD40LF, a strong increase in cytokine production occurred in B-CLL cells as well as in normal B cells. The surface molecules CD40, CD58, CD80, CD86, CD54, and MHC class I molecules were up-regulated in B-CLL cells, whereas CD95 expression was not influenced by CpG-ODN stimulation. The same pattern of surface molecule regulation was observed in normal B cells, but up-regulation of CD40 was significantly stronger in B-CLL cells. Costimulation with CpG-ODN and CD40LF resulted in further up-regulation of CD58, CD80, CD86, and MHC class I molecules. In contrast, CD95 expression induced by CD40-ligation was inhibited by CpG-ODN. CpG-ODN activated B-CLL cells acquired a strong stimulatory capacity toward T cells in allogeneic mixed lymphocyte reaction. This effect was completely inhibited by a combination of anti-CD80 and anti-CD86 monoclonal antibody. Taken together, these findings suggest the possible use of CpG-ODN for immunotherapeutic strategies in patients with B-CLL.     

FCRL2 mRNA expression is inversely associated with clinical progression in chronic lymphocytic leukemia

Fc receptor‐like 2 (FCRL2) is highly expressed on B‐cell chronic lymphocytic leukemia (B‐CLL) cells and could possibly influence disease pathogenesis. Therefore, we investigated FCRL2 mRNA expression in a large cohort with 152 CLL patients in order to assess its role in risk prediction in B‐CLL. FCRL2 mRNA expression was found to be expressed at considerably higher levels in peripheral blood mononuclear cells (PBMC) of B‐CLL patients compared to controls (range 1.35‐ to 210‐fold upregulation; P < 0.0001) and cells of other hematological diseases. Patients with high FCRL2 expression (according to ROC‐analysis) had a significantly longer treatment‐free survival (TFS) and overall survival (OS) than patients with low FCRL2 expression (median TFS: 119 vs. 34 months, P < 0.0001; median OS: 321 months vs. not reached, P = 0.009). Univariate comparisons found that FCRL2 expression was weakly associated with IGHV mutation status (P = 0.05), CD38 status (P < 0.0001) and ZAP‐70 status (P < 0.0001). Furthermore, we show that the combination of FCRL2 with ZAP70‐, CD38‐ or IGHV‐status could further significantly refine the prognostic information provided by either of the factors alone in TFS and OS. In multivariate analysis low FCRL2 expression was a significant independent prognostic factor (HR 2.4; P = 0.005). Here we demonstrate that the level of FCRL2 expression is correlated with prognosis in B‐CLL.     

Evaluation of Interleukin-9 Expression as a Potential Therapeutic Target in Chronic Lymphocytic Leukemia in a Cohort of Egyptian Patients

Chronic lymphocytic leukemia (CLL) is a common lymphoid malignancy that has a highly variable clinical course. Genomic features as zeta-chain-associated protein kinase 70 (ZAP70) expression and CD38 expression provide further differentiation of disease prognosis. Extensive studies have confirmed the oncogenic activities of IL-9 in lymphoma. The aim of the current study was to investigate the contribution of IL-9 expression to the pathogenesis of CLL and its correlation to other prognostic parameters. This study was conducted on 80 patients at diagnosis with CLL and 80 healthy controls. Using real time polymerase chain reaction and enzyme linked immunosorbant assay, IL-9 mRNA expression and its serum level were compared between patients and controls. They were both correlated with other prognostic factors. Results: There was an overexpression of IL-9 in CLL patients that correlated with modified Rai staging, ZAP70, CD38 and all hallmarks of an active and aggressive disease. The correlation between IL-9 upregulation and patient characteristics provided direct clinical evidence for its contribution to the pathogenesis of CLL. In conclusion, significantly higher expression of IL-9 measured at both the mRNA and the protein levels in patients with CLL that correlates with more complex course of the disease and worse prognosis may allow one to speculate its importance in the pathogenesis of the disease and its possible impact on prognosis.