In this study, we characterized nuclear factor B (NF- B IntroductionB-cell chronic lymphocytic leukemia (CLL) is a malignancy characterized by the accumulation of CD5, CD19, and CD23 positive lymphocytes. Diagnosis is aided by the CLL immunophenotyping score which includes assessment of CD5 and CD23, FMC7, CD79b, and surface IgM. 1 Although CLL is the commonest leukemia in the Western world, it manifests a very heterogeneous clinical course, with some patients having normal age-adjusted survival, whereas the median survival for those patients with advanced stage disease is only 3 years. 2 The factors that contribute to the pathogenesis and progression of this disease are poorly understood, but decreased susceptibility to apoptosis 3 and dysregulated proliferation have been implicated. 4 Clinical studies have shown that high ZAP-70 expression, high CD38 expression, unmutated V H genes, and cytogenetic abnormalities (especially deletions of 11q and 17p) are all associated with a poor prognosis. [5][6][7][8][9] Nuclear factor B (NF-B) is a collective name for a group of inducible homodimeric and heterodimeric transcription factors made up of members of the Rel family of DNA-binding proteins. In humans, this family is composed of c-Rel, Rel B, p50, p52, and Rel A (p65) which, when bound in the cytoplasm to inhibitor of NF-B (IB) proteins, are inactive. 10,11 Various factors, including ligation of CD40 or the B-cell receptor (BCR), result in proteosomal degradation of IB releasing NF-B, which then translocates to the nucleus. 10,11 Once in the nucleus, NF-B can enhance survival by inducing apoptosis inhibitory proteins, including inhibitor of apoptosis proteins (IAPs), Fas-associated death domain (FADD)-like interleukin (IL)-1-converting enzyme (FLICE), and FADD-like IL-1-converting enzyme-inhibitory protein (FLIP). 12-14 CLL cells have been reported to exhibit high constitutive NF-B activation compared with normal B lymphocytes. [15][16][17] Although the exact factors responsible for the constitutive expression of NF-B are not fully resolved, many factors, including Akt activation, BCR signaling, CD40 ligation, IL-4, and B-cell activating factor (BAFF), have been shown to increase NF-B activity and enhance CLL cell survival, with members of the Bcl-2 family being principal transcriptional targets. [18][19][20][21][22] Several recent studies have demonstrated the proof of concept of the effectiveness of targeting NF-B in hematologic malignancies, including CLL 23,24 and acute myeloid leukemia. 25,26 In this study, we first set out to determine the range of constitutive DNA binding of NF-B within our patient cohort and to characterize the specific subunits of NF-B in these primary CLL cells. We then went on to investigate the ability of freshly isolated CLL cells to induce NF-B expression in response to BCR Submitted November 20, 2007; accepted January 25, 2008. Prepublished online as Blood First Edition paper, January 28, 2008; DOI 10.1182 DOI 10. /blood-2007 An Inside Blood analysis of this article appears at ...
B-cell chronic lymphocytic leukaemia (CLL) cells have a differential capacity to signal through the B-cell receptor (BCR) following ligation with anti-IgM. This difference in signaling capacity has been suggested to contribute to the clinical disparity between the two subsets. However, as there appears to be no obvious correlation between response to IgM ligation and surface IgM expression, CD20 expression or CD79b expression it may be that the differential signaling response in these two distinct subsets is not caused by the BCR itself. In this study we investigated the expression of the BCR co-receptor complex (CD19, CD21 and CD81) in a cohort of CLL patients (n = 60) in order to determine whether expression of the co-receptor was associated with known CLL prognostic markers; IgVH gene mutational status, ZAP-70 and CD38 expression. In addition, we examined the relationship between co-receptor expression and the capacity of CLL cells to respond to anti-IgM cross-linking of the BCR. CLL samples showed significantly lower expression of both CD19 and CD21 when compared to normal B-lymphocytes (P <0.0001 and P<0.0001 respectively). In addition, samples derived from patients with unmutated IgVH genes showed significantly lower expression of CD19 and CD21 but not CD81 when compared to samples derived from patients with mutated VH genes (P = 0.04, P = 0.005 and P = 0.5 respectively). In contrast, none of the co-receptor molecules (CD19, CD21, CD81) were associated with CD38 expression (P = 0.82, P = 0.6, P = 0.64 respectively) or ZAP-70 expression (P = 0.2, P = 0.13 and P = 0.4 respectively). We next compared CD21 expression with levels of tyrosine phosphorylation (pre- and post-IgM ligation of the BCR). The results showed that there was no correlation between CD21 expression with either basal or IgM-stimulated tyrosine phosphorylation (P = 0.84 and P = 0.24 respectively). However, there was a significant inverse correlation with the percentage change in tyrosine phosphorylation following BCR cross-linking (P = 0.01). These data suggest that CLL cells with higher CD21 expression have a diminished magnitude of response to BCR ligation supporting the hypothesis that CLL cells with higher CD21 expression undergo receptor densitisation. Conversely, patients with lower CD21 expression may avoid receptor densitisation and consequently have a higher capacity for signaling following ligation of the BCR. Taken together, our data indicate a role for the BCR co-receptor in the signaling capacity of CLL cells and hence in the clinical course of the disease.
In this study we devised an equation that provides a new way of identifying B-cell chronic lymphocytic leukemia (CLL) patients who will require early therapeutic intervention. The equation was developed by studying the biology of the disease with a focus on cell signal transduction. Using statistical analysis, we show that measured tyrosine phosphorylation can be described as a function of VH gene mutation status, and the expression of ZAP-70 and CD38 (r2 = 0.81, n = 49). Furthermore, we show that tyrosine phosphorylation has biological relevance as it sets a threshold for the activation of the transcription factor, NF-kB, resulting in the modulation of cell survival. Using the equation derived from our training dataset we calculated tyrosine phosphorylation in a cohort of 155 unselected CLL patient samples. The patient cohort was then divided into those with calculated tyrosine phosphorylation above or below the threshold required to activate NF-kB. Patients with high calculated tyrosine phosphorylation had a shorter time to first treatment. This method identified more patients at risk of requiring early treatment than VH gene mutation status, ZAP-70 or CD38. Furthermore, calculated tyrosine phosphorylation identifies patients that will require early intervention within the mutated VH gene cohort and even patients that have Stage A disease.
We analysed tyrosine phosphorylation, NF-κB activation and survival in a cohort of 64 chronic lymphocytic leukemia patients. By graphing VH gene mutational status, CD38 expression and ZAP-70 expression as continuous variables, we investigated correlations between these prognostic markers and cell signaling. There was a strong correlation between CD38 expression and basal levels of tyrosine phosphorylation (r2 = 0.68). VH gene mutational status and ZAP-70 expression correlated with the induction of tyrosine phosphorylation by anti-IgM (r2 = 0.31 and 0.46 respectively). These findings suggest that tyrosine phosphorylation integrates these three prognostic phenotypes. In the context of NF-κB signaling, the percentage change in tyrosine phosphorylation (post anti-IgM) and ZAP-70 expression correlated with NF-κB activation (r2 = 0.2 and 0.19 respectively) and the ability to activate NF-κB was strongly correlated with cell survival (r2 = 0.67). The therapeutic rationale for the inhibition of NF-κB was strengthened by our observations that the NF-κB inhibitor, BAY 11–7082, caused a concentration-dependent increase in apoptosis in CLL samples. Taken together, our data suggest that CD38 is a surrogate marker of basal cellular activation in CLL that is independent of the B-cell receptor. Furthermore, ZAP-70 expression is the most important predictor of tyrosine phosphorylation, downstream NF-κB activation and CLL cell survival following B-cell receptor cross-linking. These findings provide a mechanism-based rationale for the poor prognosis of CD38+ and ZAP-70+ CLL patients.
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