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, or 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 strengthening 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” analysis of BCR signaling by measuring single-cell profiles of phosphoprotein 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 phosphoproteins on the route of BCR signaling, including p-Syk, p-NF-kappaB, p-Erk1/2, p-p38 and p-JNK. BCR was cross-linked by incubating cells with anti-IgM antibodies. Unsupervised clustering analysis distinguished BCR response profiles of phosphoproteins that differentiated cases of CLL with mutated IGHV from those with unmutated IGHV (P=0.0003), cases with low levels of CD38 expression from those with high levels (P=0.0004) and cases with ZAP-70-negative leukemic cells from cases that were ZAP-70-positive (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. Independent survival analysis of time to progression via fitting Cox proportional hazards models comprising clinical covariates and/or BCR network response to modulation demonstrated that measuring modulated BCR network signaling can yield improved prognostic information compared to CD38 status alone (likelihood ratio test 5.8 for CD38 versus 10.6 for signaling) and enhance prognostic assessment using IGHV status (likelihood ratio test for IGHV = 14.8 versus for IGHV + signaling = 17.9). This study shows that single-cell profiles of BCR phosphoprotein networks are associated with prognostic parameters, disease progression and overall survival in CLL.
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