Immunohistochemical analysis of IL-6, IL-8/CXCR2 axis, Tyr p-STAT-3, and SOCS-3 in lymph nodes from patients with chronic lymphocytic leukemia: correlation between microvascular characteristics and prognostic significance

Abstract

A number of studies have looked into the pathophysiological role of angiogenesis in CLL, but the results have often been inconsistent. We aimed to gain direct insight into the angiogenic process in lymph nodes involved by CLL, focusing on proangiogenic cytokines and microvessel morphometry. The tissue levels of VEGF, Th-2 cytokines IL-6 and IL-8, IL-8 receptor CXCR2, and tyrosine p-STAT-3/SOCS-3 axis modulating cytokine expression were evaluated immunohistochemically in 62 CLL/SLL cases. Microvascular characteristics were evaluated by image analysis. Results were analyzed with regard to clinicopathological characteristics. Proliferation centers (PCs) were less well vascularised compared to non-PC areas. IL-8 and CXCR2 expression was distinctly uncommon as opposed to IL-6, VEGF and SOCS-3, which were detected in the vast majority of cases. The latter two molecule expressions were more pronounced in the PCs in ∼ 40% of the cases. p-STAT-3 immunoreactivity was recorded in 66.67% of the cases with a predilection for PCs. Microvessel morphometry was unrelated to proangiogenic cytokines, p-STAT-3, SOCS-3, or survival. Microvascular caliber and VEGF expression were higher in Binet stage A, whereas IL-6 expression was higher in stage C. VEGF and p-STAT-3 exerted a favorable effect on progression, which remained significant in multivariate analysis, thereby constituting potential outcome predictors in CLL patients.

Figure 1

(a) Immunohistochemical staining of CD34 in a CLL case. (b) Same field as in (a). The outline of each vessel is traced; the red layer represents the section area of each vessel outside the PCs, whereas the yellow layer represents the section area of each vessel within the PCs. PCs display clearly higher MVD and TVA and rounder vessels when compared to non-PC areas. (PC: proliferation centers).

Figure 2

Box plots illustrating the lower levels of MVD, major axis length, minor axis length, area, perimeter, and TVA and the higher levels of shape factor and VEGF H-score in the PCs when compared to the non-PC areas.

Figure 3

Immunohistochemical expression of IL-8 (a), CXCR2 (b), IL-6 (c), tyrosine p-STAT-3 (d, e), SOCS-3 (f), and VEGF (g, h) in lymph nodes from CLL patients. (a) A CLL case with very few scattered IL-8 positive lymphoid cells. The inset shows an IL-8 positive cell in a higher magnification. Note the positive endothelial cells. (b) CXCR2 expression in a CLL lymph node. (c) IL-6 expression homogeneously distributed throughout the lymphoid tissue. (d) Tyrosine p-STAT-3 in a case showing a more pronounced immunoreactivity in the PCs. Note that endothelial cells are strongly positive. (e) Scattered tyrosine p-STAT-3 positive lymphoid cells along with positive endothelial cells in a CLL case. (f) Higher SOCS-3 expression in the PC in a CLL case. (g) (h) Pronounced VEGF immunoexpression in the PCs compared with the area outside the PCs. Higher magnification (h) showing the cytoplasmic immunoreactivity of VEGF. (PC: proliferation centers; E: endothelial cells).

Figure 4

Box plot illustrating the correlation between IL-6 H-score and Binet stage. IL-6 was higher in stage C, followed by stage A, whereas stage B cases displayed the lower levels of IL-6 H-score. Post hoc analysis indicated that the only significant difference was that between stages B and C (P = 0.0471, Tukey HSD method).

Figure 5

(a, b) Kaplan Meier survival curves for OS according to tyrosine p-STAT-3 immunoreactivity (a) and VEGF H-score (b). (c) Kaplan Meier failure curves for TFT according to VEGF H-score. (d, e) Kaplan Meier failure curves for TTP according to tyrosine p-STAT-3 immunoreactivity (d) and VEGF H-score (e).