Phosphorylation profile of CD5^high/CXCR4^low leukemic cells in patients with CLL treated with ibrutinib. Cell sorting purified CD19⁺/CD5^high/CXCR4^low cells obtained from PBMC of 3 patients with CLL before and after 4 weeks of ibrutinib therapy were used to perform a phosphoarray analysis. (A) Volcano plot showing the fold change vs the P value obtained for each evaluated phosphorylation site. Black dots refer to sites that did not change after treatment (63 phosphorylation sites). Depicted in color are the sites that were significantly dephosphorylated (green dots, 12 sites) or phosphorylated (red dots, 19 sites) during therapy (P < .05; n = 3; 2-tailed paired Student t test). The reference for each numbered dot is depicted in panel B. (B) Heat map showing the targeted proteins with their corresponding phosphorylation sites in 3 CLL patient samples. Heat map scale is shown at the bottom. (C) Statistically significant changes in phosphorylation rate (red, upper panel) or dephosphorylation rate (green, lower panel) during ibrutinib administration are shown. Each square represents the signal ratio of the antibody against the unphosphorylated form of the site before and after treatment, connected with the round dot for the signal ratio of the phosphorylated form (P < .05; n = 3; 2-tailed paired Student t test). Relevant proteins associated with inactivation of PI3K/AKT, JAK/STAT, and p53 pathway are indicated in bold at the x axes.