Ibrutinib induces rapid and sustained inhibition of BCR and NF-κB signaling in vivo in circulating CLL cells. (A-C) Change in BCR and NF-κB signature scores (identified in reference 1 and described in Materials and methods) in purified CLL cells on treatment. (A) Mean (± SEM) percent reduction in signature scores (n = 8 patients, in whom all time points were available). Comparisons by paired Student t test: *P < .05, **P < .01, and ***P < .001. (B) Percent reduction on day 28 compared with pretreatment. Each dot represents a different patient (n = 25). The line represents the median. (C) Correlation of reduction in BCR and NF-κB signature scores. Each dot represents one patient. R and P values of Pearson correlation are displayed (n = 25). (D-E) PBMCs were fixed, permeabilized, and stained with the indicated antibody. Results shown are for the CLL population (CD5⁺/CD19⁺). (D) A representative histogram of pPLCγ2 staining. Isotype control is represented by the gray shaded area, the dashed line represents pretreatment, and the solid black line day 28. (E) The mean (± SEM) percent of pPLCγ2 and pERK expressing CLL cells pretreatment (Pre) and on day 28 of ibrutinib treatment is shown (n = 30). (F) Quantification of nuclear p50 was done on nuclear lysates from CLL cells pretreatment (Pre) and on day 28 of ibrutinib treatment (n = 10).