Peritumoral lymphangiogenesis induced by vascular endothelial growth factor C and D promotes lymph node metastasis in breast cancer patients

Abstract

Background: Mounting clinical and experimental data suggest that the migration of tumor cells into lymph nodes is greatly facilitated by lymphangiogenesis. Vascular endothelial growth factor (VEGF)-C and D have been identified as lymphangiogenic growth factors and play an important role in tumor lymphangiogenesis. The purpose of this study was to investigate the location of lymphangiogenesis driven by tumor-derived VEGF-C/D in breast cancer, and to determine the role of intratumoral and peritumoral lymphatic vessel density (LVD) in lymphangiogenesis in breast cancer.

Methods: The expression levels of VEGF-C/D were determined by immunohistochemistry, and intratumoral LVD and peritumoral LVD were assessed using immunohistochemistry and the D2-40 antibody in 73 patients with primary breast cancer. The associations of intratumoral LVD and peritumoral LVD with VEGF-C/D expression, clinicopathological features and prognosis were assessed.

Results: VEGF-C and D expression were significantly higher in breast cancer than benign disease (P < 0.01). VEGF-C (P < 0.001) and VEGF-D (P = 0.005) expression were significantly associated with peritumoral LVD, but not intratumoral LVD. Intratumoral LVD was associated with tumor size (P = 0.01). Peritumoral LVD was significantly associated with lymph node metastasis (LNM; P = 0.005), lymphatic vessel invasion (LVI; P = 0.017) and late tumor,node, metastasis (TNM) stage (P = 0.011). Moreover, peritumoral LVD was an independent risk factor for axillary lymph node metastasis, overall survival and disease-free survival in multivariate analysis.

Conclusions: This study suggests that tumor-derived VEGF-C/D induce peritumoral lymphangiogenesis, which may be one mechanism that leads to lymphatic invasion and metastatic spread. Peritumoral LVD has potential as an independent prognostic factor in breast cancer patients.

Figure 1

Immunohistochemical analysis of VEGF-C and VEGF-D expression in primary breast carcinoma. (A) Histopathology by hematoxylin-eosin staining of breast cancer; (B) Strong expression of VEGF-C and (D) negative control in breast cancer; (C) Strong expression of VEGF-D and (E) negative control in breast cancer. Diffuse, strong positive VEGF-C (B) and VEGF-D (C) immunostaining was mainly observed in the cytoplasm of breast cancer cells (magnification × 400). VEGF-C/D, vascular endothelial growth factor C /D.

Figure 2

Immunohistochemical analysis of lymphatic vessels in primary breast carcinoma. (A) Intense, specific D2-40 immunoreactivity was only observed in lymphatic endothelial cells. The intratumoral lymphatic vessels are small, irregular and collapsed (arrow). (B) The peritumoral lymphatic vessels located at the invasive edge of tumors are frequent, often large and dilated (arrow); magnification × 200.(C) Immunohistochemical visualization of invading breast cancer cells in the lymphatic vessels of the peritumoral region of a primary breast carcinoma. The black arrow indicates lymphatic vessel invasion (LVI); magnification × 200.

Figure 3

Kaplan-Meier disease-related overall survival (A and C) and disease-free survival (B and D) curves; stratified by low versus high intratumoral LVD (A and B) and low versus high peritumoral LVD (C and D) in invasive ductal breast carcinoma. The median LVD values were used as cutoff values. LVD, lymphatic vessel density.