SINGLE-CELL PROFILES OF B-CELL RECEPTOR PHOSPHO-PROTEIN NETWORKS ASSOCIATED WITH PROGNOSIS AND PROGRESSION 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 with- in the immunoglobulin variable heavy chain genes (IGHV), the expres- sion 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 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 net- works 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 phos- phoproteins on the route of BCR signaling, including p-Syk, p-NF-kap- paB, p-Erk1/2, p-p38 and p-JNK. BCR was cross-linked by incubating cells with anti-IgM antibodies. The unsupervised clustering analysis dis- tinguished BCR response profiles of phospho-proteins that differentiat- ed 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. This study shows that single-cell profiles of BCR phospho-protein networks are associated with prognos- tic parameters, disease progression and overall survival in CLL. (This study was supported by: Regione Veneto “Ricerca Sanitaria Finalizzata”; “Fondazione G. Berlucchi per la Ricerca sul Cancro”; AIRC - Associ- azione Italiana Ricerca sul Cancro; Fondazione CARIVERONA and Fon- dazione CARIPARO)