Molecular determinants of B-cell anergy. (A) ERK1/2 constitutive phosphorylation significantly correlated with NF-ATc1 nuclear translocation (P = .004). Constitutive ERK1/2 and NF-ATc1 activation (each normalized to specific positive controls) were evaluated in a cohort of 52 freshly purified samples. Samples were grouped on the basis of the ERK1/2 phosphorylation status [23 pERK(+), with relative % pERK >10%, and 29 pERK(−), with relative % p-ERK <10%], and the percentage of NF-ATc1 activation was reported on the y-axis. (B) ERK1/2 activation and calcium flux. Leukemic cells from 32 patients were labeled with the calcium-sensitive dye Fluo3-AM and analyzed by flow cytometry before and after addition of F(ab′)₂ anti-IgM. Samples were grouped on the basis of the ERK1/2 activation status. Graph shows individual data point and means (horizontal line). Data were analyzed by using Mann-Whitney U test (P value is indicated). (C) ERK1/2 activation and IgM expression. Leukemic cells from 24 patients were analyzed for surface IgM expression (mean fluorescence intensity) and grouped on the basis of pERK1/2 status. The graph shows individual data point and means (horizontal line). Data were analyzed with the Mann-Whitney U test (P value is indicated). (D) Anergic cells displayed an increasing survival in vitro. Anergic signature correlated with increased in vitro survival of CLL cells. Freshly purified leukemic cells (10 pERK(+) and 9 pERK(−) samples) were cultured for 24 or 48 hours, and cell viability was analyzed with Annexin/PI staining. Samples were grouped on the basis of the ERK1/2 activation status, and data were analyzed with the Mann-Whitney U test (P value is indicated).