Flow cytometry analysis of B-cell receptor phospho-protein networks in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) patients exhibit a variable clinical course. Several biological parameters have been shown to be associated with clinical outcome in CLL. Among them, the most reliable markers are represented by the absence of somatic mutations within the immunoglobulin variable heavy chain genes (IGHV), the expression of CD38 antigen, the presence of the ZAP-70 tyrosine kinase. These parameters of poor clinical outcome are structurally and/or functionally linked to B-cell Receptor (BCR) expressed by CLL cells, thereby strengthen the hypothesis that antigenic stimulation mediated by the BCR represents a driving event in the onset and progression of the malignant B cells. To investigate whether different BCR signaling networks may distinguish clinical-biological groups of CLL patients, we applied a “network level” view of BCR signaling by analyzing single-cell profiles of phospho-protein networks by flow cytometry. We evaluated the response to BCR engagement in primary cells isolated from 27 CLL patients by analyzing the phosphorylation states of 5 phospho-proteins on the route of BCR signaling, which included p-Syk, p-NF-kappaB, p-Erk1/2, p-p38 and p-JNK. BCR was cross-linked by incubating cells with anti-IgM antibodies. The unsupervised clustering analysis distinguished BCR response profiles of phospho-proteins that differentiated cases of CLL with mutated IGHV from those with unmutated IGHV (p=0.0003), cases with low levels of CD38 from those with high levels (p=0.0004) and cases with low levels of ZAP-70 from those with high levels (p=0.001). Furthermore, the same BCR response profiles were also associated with time to progression (p=0,0014) and with overall survival (p=0,049), as assessed by Kaplan–Meier curves and the log-rank test.This study shows that single-cell profiles of BCR phospho-protein networks are associated with prognostic parameters and disease progression in CLL