Angiogenesis and hypoxia in lymph node metastases is predicted by the angiogenesis and hypoxia in the primary tumour in patients with breast cancer

Abstract

Hypoxia and angiogenesis are important factors in breast cancer progression. Little is known of hypoxia and angiogenesis in lymph node metastases of breast cancer. The aim of this study was to quantify hypoxia, by hypoxia-induced marker expression levels, and angiogenesis, by endothelial cell proliferation, comparing primary breast tumours and axillary lymph node metastases. Tissue sections of the primary tumour and a lymph node metastasis of 60 patients with breast cancer were immunohistochemically stained for the hypoxia-markers carbonic anhydrase 9 (CA9), hypoxia-inducible factor-1alpha (Hif-1alpha) and DEC-1 and for CD34/Ki-67. Endothelial cell proliferation fraction (ECP%) and tumour cell proliferation fraction (TCP%) were assessed. On haematoxylin-eosin stain, the growth pattern and the presence of a fibrotic focus were assessed. Hypoxia-marker expression, ECP% and TCP% in primary tumours and in lymph node metastases were correlated to each other and to clinico-pathological variables. Median ECP% and TCP% in primary tumours and lymph node metastases were comparable (primary tumours: ECP%=4.02, TCP%=19.54; lymph node metastases: ECP%=5.47, TCP%=21.26). ECP% correlated with TCP% (primary tumours: r=0.63, P<0.001; lymph node metastases: r=0.76, P<0.001). CA9 and Hif-1alpha expression were correlated (primary tumours P=0.005; lymph node metastases P<0.001). In primary tumours, CA9 and Hif-1alpha expression were correlated with DEC-1 expression (P=0.05), presence of a fibrotic focus (P<0.007) and mixed/expansive growth pattern (P<0.001). Primary tumours and lymph node metastases with CA9 or Hif-1alpha expression had a higher ECP% and TCP% (P<0.003); in primary tumours, mixed/expansive growth pattern and fibrotic focus were characterised by higher ECP% (P=0.03). Furthermore, between primary tumours and lymph node metastases a correlation was found for ECP%, TCP%, CA9 and Hif-1alpha expression (ECP% r=0.51, P<0.001; TCP r=0.77, P<0.001; CA9 and Hif-1alpha P<0.001). Our data demonstrate that the growth of breast cancer lymph node metastases is angiogenesis dependent and that angiogenesis and hypoxia in the primary tumour predict angiogenesis and hypoxia in the lymph node metastases. Together with previous findings in breast cancer liver metastases, which grow in 96% of cases angiogenesis independently, these data suggest that both the intrinsic growth characteristics and angiogenic potential of breast cancer cells and the site-specific tumour microenvironment determine angiogenesis and hypoxia in breast cancer.

Figure 1

Overview of the histological and immunohistochemical stainings used, in a primary tumour (left: A, C and E) and in the corresponding lymph node metastasis (right: B, D and F) with expression of hypoxia markers and high ECP%. A and B: HE staining showed the presence of a large fibrotic focus (^*) in the primary tumour. C and D: On CD34/Ki-67 IHC double-staining ECP% was high. Insets show Ki-67-positive (brown nucleus) endothelial cells (red cytoplasm) (black arrows). E and F: Membranous CA9 staining of tumour cells in both the primary tumour and in the lymph node metastasis was demonstrated. Insets show nuclear Hif-1α (upper inset) and DEC-1 (lower inset) expression in the same tumour region. ECP%: endothelial cell proliferation fraction; HE: haematoxylin–eosin; CA9: carbonic anhydrase 9; Hif-1α: hypoxia-inducible factor-1α; DEC-1: differentiated embryo-chondrocyte-expressed gene 1.

Figure 2

Scatter plot showing a strong correlation between ECP% and TCP% in both primary tumours (left) and lymph node metastases (right). ECP%: endothelial cell proliferation fraction; TCP%: tumour cell proliferation fraction; PT: primary tumour; LNM: lymph node metastasis.

Figure 3

Box and Whiskers plot of ECP% (left, black) and TCP% (right, grey) in CA9, DEC-1 and Hif-1α-positive (Pos) and -negative (Neg) primary tumours (A) and lymph node metastsases (B). Primary tumours and lymph node metastases with CA9 or Hif-1α expression had significantly higher ECP% and TCP%. No difference in ECP% or TCP% was found between primary tumours and lymph node metastases with or without DEC-1 expression. PT: primary tumour; LNM: lymph node metatasis; ECP%: endothelial cell proliferation fraction; TCP%: tumour cell proliferation fraction; CA9: carbonic anhydrase 9; Hif-1α: hypoxia inducible factor-1α; DEC-1: differentiated embro-chondrocyte-expressed gene 1.

Figure 4

Scatter plot representing the correlation between primary tumours and lymph node metastases for ECP% (left) and TCP% (right). A strong correlation was found for both TCP% and ECP%. ECP%: endothelial cell proliferation fraction; TCP%: tumour cell proliferation fraction; PT: primary tumour; LNM: lymph node metastasis.