Lymphatic Expression of CLEVER-1 in Breast Cancer and Its Relationship with Lymph Node Metastasis

BACKGROUND Mechanisms regulating breast cancer lymph node metastasis are unclear. Staining of CLEVER-1 (common lymphatic endothelial and vascular endothelial receptor-1) in human breast tumors was used, along with in vitro techniques, to assess involvement in the metastatic process. METHODS 148 sections of primary invasive breast cancers, with 10 yr follow-up, were stained with anti-CLEVER-1. Leukocyte infiltration was assessed, along with involvement of specific subpopulations by staining with CD83 (mature dendritic cells, mDC), CD209 (immature DC, iDC) and CD68 (macrophage, Mϕ). In vitro expression of CLEVER-1 on lymphatic (LEC) and blood endothelial cells (BEC) was examined by flow cytometry. RESULTS In vitro results showed that although both endothelial cell types express CLEVER-1, surface expression was only evident on LEC. In tumour sections CLEVER-1 was expressed in blood vessels (BV, 61.4% of samples), lymphatic vessels (LV, 18.2% of samples) and in Mϕ/DCs (82.4% of samples). However, only CLEVER-1 expression in LV was associated with LN metastasis (p = 0.027) and with Mϕ indices (p = 0.021). Although LV CLEVER-1 was associated with LN positivity there was no significant correlation with recurrence or overall survival, BV CLEVER-1 expression was, however, associated with increased risk of recurrence (p = 0.049). The density of inflammatory infiltrate correlated with CLEVER-1 expression in BV (p < 0.001) and LV (p = 0.004). CONCLUSIONS The associations between CLEVER-1 expression on endothelial vessels and macrophage/leukocyte infiltration is suggestive of its regulation by inflammatory conditions in breast cancer, most likely by macrophage-associated cytokines. Its upregulation on LV, related surface expression, and association with LN metastasis suggest that it may be an important mediator of tumor cell metastasis to LN.