Increased AKT activation in CLL subsets associated with RT and increased frequency in patients with RT. (A) Flow diagram of AKT activation screening setup. (B) Histogram showing pAKT (Ser473)/panAKT expression (AKT activation) from freshly isolated peripheral B cells, in a cohort of patients with CLL stratified according to mutations in NOTCH1, TP53, SF3B1, and ATM (n = 46). Colored dots represent chromosomal aberrations identified via fluorescence in situ hybridization (blue = trisomy12; red = 17p deletion; green = 11q deletion; yellow = 13q deletion). (C) Immunofluorescence imaging of CLL (n = 8), RT (n = 19), and DLBCL (n = 12) for pAKT (Ser473). Proportion of cases defined as negative (black), positive (pink), or double positive (red) shown as pie charts for each entity (upper panel). Representative images from positive and negative cases from each entity (lower panel). All images shown at ×40 magnification. Nuclear staining (4′6-diamidino-2-phenylindole) and pAKT (Ser473) staining shown in orange. Significant differences in AKT activation between wild-type and TP53/NOTCH1/SF3B1/ATM mutated CLL peripheral blood mononuclear cells (PBMCs) by immunoblotting assessed by using one-way analysis of variance, with Tukey multiple comparison correction.