Intracellular ERK Phosphorylation Levels after Ligation of the B Cell Receptor Are Associated with CD38 and Immunoglobulin Gene (V_H) Germline Concordance in Chronic Lymphocytic Leukaemia

Clinically useful prognostic markers are well described in chronic lymphocytic leukaemia (CLL) and include cell surface expression of CD38 and levels of the intracellular kinase ZAP70. The variable hinge region of the immunoglobulin heavy chain gene (V_H) mutation status is well described and highly prognostic but at present remains primarily a research tool. The presence or absence of activation of intracellular signalling cascades by phosphorylation in response to cross linking of the B cell receptor (BCR) in vitro has also been shown to carry prognostic merit in CLL in a number of studies. These studies have used western blotting based approaches making screening of large sample numbers time consuming.

We investigated the correlation between intracellular kinase signalling and CD38, ZAP70 and V_H status using a novel ELISA assay for phospho Extracellular signal Regulated Kinase (pERK). CLL cells from 101 patients were either stimulated by BCR cross-linking with goat anti-human IgM Fc5μ fragment or treated with Goat IgG F(ab’) fragment as an isotype control or treated with Phorbol-12-Myristate-13-Acetate (PMA) as a positive control. All treatments were given for a 15 minute incubation prior to cell lysis and protein extraction.

We validated our sample pool with analysis of cumulative survival versus V_H mutation status. Using a 98% deviation from germline as the unmutated : mutated cut off this confirmed a significant survival advantage in the mutated cases p=0.011 (Mantel-Cox log rank, unmutated mean survival 3209 days vs. mutated mean survival 7569 days). For the analysis of signalling status samples demonstrating a greater than 2 fold increase in absolute pERK levels over isotype were designated as signallers. Of 101 samples analysed 33 (33%) were signallers. Mean survival for signallers was 2826 days whereas for non-signallers it was 7059 days. However this difference was not significant p=0.404 (Mantel-Cox log rank).

A significant positive correlation between signaller status and V_H concordance to germline was demonstrated (p=0.014, Mann Whitney test; Pearson correlation coefficient 0.226, p=0.023). CD38 positivity and pERK signalling status also showed a significant relationship (p=0.003, Chi Squared test; Pearson correlation coefficient 0.364, p=0.002). The relationship between pERK and ZAP70 did not achieve significance (p=0.054, Chi Squared test; Pearson correlation coefficient 0.211, p=0.039).

Taken as a whole these data provide further strong evidence that retention of the ability to signal across the BCR is important in CLL prognosis. We have demonstrated that ELISA is an highly efficient and fully quantitative tool for such analysis. ELISA also facilitates the study of larger sample numbers in comparison to more traditional techniques. We are continuing to use ELISA on different targets as an effective way of progressing the study of intracellular signalling. This study of intracellular signalling remains a valid and important area of translational research in CLL.