Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer

Abstract

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.

Figure 1

Membranous/cytoplasmic Dll4 expression (brown) in endothelium adjacent to breast cancer (A) with matching CD31 expression (B) is shown. Endothelium adjacent to normal breast does not express Dll4 by immunohistochemistry (C), but is identified by CD31 (D). Scale bar = 100 μm. Representative examples of Dll4 scoring in endothelial cells (arrowheads), scored 0 (E), 1 (F), and 2 (G) are shown. Scale bar = 25 μm.

Figure 2

Kaplan-Meier overall survival (A) and relapse-free survival (B) curves for breast cancer patients subgrouped according to Dll4 expression is shown. Overall survival, Cox regression: score 2 vs. score 0; hazard ratio: 2.29 (95% confidence interval, 1.37 to 3.82), P = 0.002; score 2 vs. score 1, hazard ratio: 2.54 (95% confidence interval, 1.62 to 3.98), P = 0.00005. Relapse-free survival, Cox regression: score 2 vs. score 0; hazard ratio: 2.03 (95% confidence interval, 1.19 to 3.47), P = 0.01; score 2 vs. score 1, hazard ratio: 2.09 (95% confidence interval, 1.30 to 3.36), P = 0.002.