Quantification of S100-A9 expression in the leukemic clone. Flow cytometry analysis of PBMCs was performed evaluating IgM, CD5, and S100-A9⁺ cells between HDs and the different CLL subgroups. CD19⁺ cells of age-matched HDs were used to obtain reference values, and control isotypes were used for IgM and S100-A9 antibodies. (A) S100-A9 expression in indolent and progressive CLL samples during disease progression. Flow cytometry analysis was performed with specific antibodies, as described in supplemental Table 1. Five age-matched HDs to determine the physiological levels of S100-A9 in B lymphocytes and a total of 16 indolent CLLs and 18 progressive CLLs at the time of progression were evaluated. A significant increase of S100-A9 levels was found in the samples obtained from Prog-ddp CLLs (mean, 32.44) compared with samples from indolent CLLs (mean, 14.98; 1-way analysis of variance [ANOVA], P < .01). No significant changes were observed between HD samples compared with indolent CLLs (mean, 5.55 and 14.98, respectively). ns, not significant. (B) S100-A9 protein expression levels in B lymphocytes at different times of disease evolution. Five HDs, 8 Ind-4years, and 8 progressive CLLs at the time of onset and 8 during disease progression were evaluated. A significantly higher level of S100-A9 protein in the leukemic cells from the Prog-ddp samples (mean, 42.50) compared with Prog-dt (mean, 23.24) and with Ind-4years (mean, 14.64) was demonstrated (1-way ANOVA, P < .01). No significant differences were visualized comparing the expression of S100-A9 in B lymphocytes (mean, 1.38) with Ind-4years (mean, 14.64) or Prog-dt (mean, 23.24). Microscopy analysis was performed with specific anti-IgM, anti-CD5, and anti–S100-A9 (supplemental Table 1). (C) S100-A9 expression in the leukemic clone was confirmed as a discrete cytoplasmic pattern shown in red color. DNA staining was performed with methyl green as described by Oppezzo et al.²²  Gray and white asterisks indicate low and high S100-A9 expression levels, respectively. Scale bar, 5μm (lower right). (D) NF-κB pathway is more activated in the samples of progressive CLLs during disease progression. Characterization of NF-κB pathway activation by immunoblot assays evaluating IκB-α and inhibitory IκB-α phosphorylation in PBMCs of the different subgroups (Prog-dt and Prog-ddp). Representative cases (10 of 20 analyzed patients) as depicted after immunoblot reaction. (E) Increased NF-κB activation in the Prog-ddp subgroup was evidenced after quantification of p65 nucleus/cytoplasm stain ratio from a total of 100 CLL cells (IgM⁺/CD5⁺) in 5 samples from each subgroup (Prog-dt and Prog-ddp; n = 10). Prog-dt samples display a low percentage of cells with the transcription factor p65 in the nucleus (mean, 30) compared with Prog-ddp samples of the same patient (mean, 50; Mann-Whitney test, P = .019). Immunofluorescent staining was performed with specific human anti-p65 transcription factor, specific anti-IgM, and anti-CD5 antibodies. (F) Positive correlation of S100-A9 expression with IκB-α phosphorylation in patients with CLL. The relationship between S100-A9 protein expression and IκB-α phosphorylation was investigated. A significant positive correlation was detected (r² = 0.35; Spearman’s rank test, P < .001).