Comparison of molecular determinants of angiogenesis and lymphangiogenesis in lymph node metastases and in primary tumours of patients with breast cancer

Abstract

Angiogenesis and lymphangiogenesis are complex processes, driven by multiple factors. In primary breast tumours (PTs), VEGFA, -C and -D are the most important (lymph)angiogenic factors. The induction of lymphangiogenesis in axillary lymph node (LN) metastases of patients with breast cancer was described recently. To compare the molecular determinants of (lymph)angiogenesis in LN metastases and PTs of breast cancer patients, RNA was isolated from formalin-fixed, paraffin-embedded tissue sections of a metastatically involved and uninvolved LN and the PT from 26 lymph node-positive patients. The expression of 12 (lymph)angiogenic markers was measured by qRT-PCR. Expression was correlated with tumour cell proliferation, angiogenesis and lymphangiogenesis, quantified by tumour cell proliferation fraction (TCP%) and (lymphatic) endothelial cell proliferation fraction [(L)ECP%]. TCP%, ECP% and LECP% were assessed on immunohistochemical double stains for CD34/Ki-67 and D2-40/Ki-67, respectively. In involved LNs, the relative gene expression levels of PROX1 (p < 0.001) and FGF2 (p = 0.008) were decreased and the expression levels of VEGFA (p = 0.01) and PDGFB (p = 0.002) were increased compared to uninvolved LNs. The expression of most markers was increased in PTs compared to involved LNs. In metastatically involved LNs, the expression of VEGFA correlated with ECP% (r = 0.54, p = 0.009) and LECP% (r = 0.76, p < 0.001). In PTs, VEGFA correlated only with ECP% (r = 0.74, p < 0.001). VEGFD correlated with peritumoural LECP% (r = 0.61, p = 0.001) and with VEGFC (r = 0.78, p < 0.001). Linear regression analysis confirmed the expression of VEGFA as an independent predictor of ECP% in both PTs and LN metastases and of LECP% in LN metastases. The expression of VEGFD, but not of VEGFA, independently predicted peritumoural LECP% in PTs. Our results confirm existing data that, in PTs, angiogenesis and lymphangiogenesis are respectively driven by VEGFA and VEGFD. In contrast, in LN metastases, both processes seem to be driven by VEGFA. Lymphangiogenesis in PTs and in LN metastases might thus be driven by different factors.