Analysis of AID expression in B cells from ibrutinib-treated patients with CLL. PBMC samples from patients with CLL before and after 1 and 4 weeks of ibrutinib in vivo therapy were studied by flow cytometry to measure intracellular AID protein. Leukemic cells were discriminated by gating lymphocytes and by labeling CD19 plus CD5. Expression of AID as percentage of positive cells in the proliferative CD19⁺/IgM⁺/IgG⁺ subset compared with IgM⁺/IgG^- quiescent cells or from proliferative CD19⁺/CD5^high/CXCR4^low vs CD19⁺/CD5^low/CXCR4^high quiescent cells for treatment at 3 times. (A) Results for the IgM⁺/IgG⁺ subset in the whole group of patients evaluated (left graphic) and 1 representative cytometry data (right graphic). The corresponding median values were 7.2% pre- and 2.5% at week 4 posttreatment (mean difference, 4.7%; 95% CI, 2.3-7.2; P = .0007, 1-way ANOVA, multiple comparisons test). Nonsignificant differences were found between pre- and 1 week posttreatment samples (P = .810). Right panel shows representative dot plots of the gates constructed to select the subsets and from where the histograms of AID expression were obtained. PF and QF are depicted in green and red, respectively. (B) Results for the CXCR4 low/CD5 high fraction in the whole group of patients evaluated (left graphic) and 1 representative cytometry data (right graphic). Significant differences were found comparing pr-treatment mean (10%) vs mean posttreatment at week 1, 4.5%; (mean difference, 5.68%; 95% CI, 0.91-10.45; P = .278) and vs week 3, 1.13% (mean difference, 9.5%; 95% CI, 4.3%-13.8%; P = .0016 by 1-way ANOVA, multiple comparisons test). PF and QF are depicted in green and red, respectively. (C-D) MFI AID expression on the quiescent and proliferative subsets CD19⁺IgM⁺IgG⁺ compared with IgM⁺/IgG^- quiescent cells or from proliferative CD19⁺/CD5^high/CXCR4^low vs CD19⁺/CD5^low/CXCR4^high quiescent cells, at the points indicated. Intracellular staining of AID as MFI was evaluated. Shown are representative dot plots and gate criteria (higher graphics), as well as AID histograms of cytometric MFI shifts with treatment (lower left graphic) for a single patient. The statistics of the whole cohort evaluated are shown in the lower right graphics. The mean of cells expressing AID in IgM⁺/IgG⁺ PF at pretreatment was 9.7 MFI vs 6.0 MFI at week 4 (mean differences, 3.7; 95% CI, 1.4-6.0; P = .0004; n = 10). For CXCR4^lowCD5^high PF pretreatment, 7.0 MFI vs 4.5 at week 4 MFI (mean differences, 2.4; 95% CI, 1.0-3.7; P = .0004; n = 5). One-way ANOVA with Tukey's multiple comparisons test was used in all cases (***P < .0005).