Histomorphometrical and quantitative molecular comparison of the first steps of metastasis of the primary tumour and loco-regional and distant metastases in breast cancer

Although loco-regional spread and recurrence of breast cancer can be debilitating, metastasis to distant organs is the leading cause of breast cancer-related death. One of the very early steps in the metastastic cascade is (lympho)vascular invasion, or the penetration of tumour cells into lymph and/or blood vessels in and around the primary tumour. The purpose of this project is to study different aspects of and processes involved in these very first steps of breast cancer metastasis in the primary tumour and compare them to loco-regional axillary lymph node metastases and distant metastases (e.g. liver) in patients with breast cancer.

Due to the characterization of lymphatic endothelial specific markers it is now possible to distinguish between blood and lymph vessel invasion in human cancer resection specimen and to study the contribution of both processes to breast cancer metastasis. Therefore we developed a histomorphometrical technique using immunohistochemical stains with different endothelial antibodies on serial sections of breast cancer resection specimens. Immunohistochemical detection of lymphovascular invasion was much more sensitive than detection on haematoxylin-eosin slides. About 20-25% of peritumoral and 70-75% of intratumoral lymphovascular invasion was missed on haematoxylin-eosin slides. Recently lymphovascular invasion has been added to the St.-Gallen criteria for risk assessment and treatment of patients with operable breast cancer. In the St.-Gallen criteria lymphovascular invasion is detected on haematoxylin-eosin slides. Our data suggest that immunohistochemical detection of lymphovascular invasion might be of clinical value.

Furthermore, this technique allows us to discriminate between blood and lymph vessel invasion. We were able to demonstrate that lymph vessel invasion is far more frequent in breast cancer than blood vessel invasion. We also found substantial differences between the size and extent of lymph and blood vessel invasion: lymphatic tumour cell emboli were larger and more frequent than blood vessel tumour cell emboli. We are now investigating the presence of different aerobic and anaerobic metabolic pathways in intraparenchymal tumour cells and intravascular tumour cell emboli. Recently, it has been shown that in colo-rectal tumour tissue tumour cells express proteins involved in anaerobic metabolism where tumor associated fibroblasts and endothelial cells express proteins involved in anaerobic metabolism. Both tumour compartments contribute to a harmonious metabolic domain enabling tumour cell survival and growth. Whether the same process are involved in survival of tumour cells in in intravascular tumour cell emboli and at distant metastatic sites remains to be elucidated

Another histological parameter associated with angiogenesis, lymphangiogenesis and lymphovascular invasion is the presence of a fibrotic focus. The fibrotic focus is defined as a scar-like area, consisting of fibroblasts and collagen fibres, that occupies various percentages of the center of an invasive (ductal) carcinoma of the breast. We and others have extensively demonstrated that the presence of a fibrotic focus is correlated to the presence of hypoxia, increased angiogenesis and lymphangiogenesis and lymphovascular invasion. The presence of a fibrotic focus in primary breast carcinoma’s is also associated with a poor prognosis. In collaboration with the University of Rotterdam we are currently investigating genome-wide gene expression patterns associated with the presence of a fibrotic focus.

We also studied angiogenesis and hypoxia at different tumour sites of patients with breast cancer. Endothelial cell proliferation fraction, a histomorphometrical measure of angiogenesis, tumour cell proliferation fraction and the expression of hypoxia inducible factor-1 alpha (Hif-1alpha) and one of its down stream targets -carbonic anhydrase 9 (CA9)- were correlated in lymph node metastases and in primary tumours. We concluded that tumour growth in axillary lymph node metastases, as in primary breast tumours, is angiogenesis-dependent and that angiogenesis and hypoxia in lymph node metastases is predicted by the primary tumour. This is in contrast with our previous results in liver metastases of patients with breast cancer. Apparently more than 90% of breast cancer liver metastases grow according to a non-angiogenic replacement pattern in which tumour cells replace the hepatocytes at the tumour-liver interface while preserving the existing sinusoidal liver architecture and without induction of a desmoplastic stromal reaction. Endothelial cell proliferation fraction and CA9 expression are low in these liver metastases, sustaining the hypothesis that the growth of these liver metastases is angiogenesis-independent. These results corroborate the idea that the growth of breast cancer deposits differs among different sites. The different degrees of angiogenesis-dependency might be important when considering the use of anti-angiogenic therapies for patients with primary and metastatic breast cancer. We are currently also investigating the presence and extent of lymphangiogenesis at different metastasis sites. Preliminary data suggest that in and around breast cancer lymph node metastases lymphangiogenesis is induced.

Relevant publications:
• Van den Eynden GG, Vandenberghe MK, van Dam PJ, Colpaert CG, van Dam P, Dirix LY, Vermeulen PB, Van Marck EA. Increased sentinel lymph node lymphangiogenesis is associated with nonsentinel axillary lymph node involvement in breast cancer patients with a positive sentinel node. Clin Cancer Res. 2007 Sep 15;13(18 Pt 1):5391-7.
• Van den Eynden GG, Van der Auwera I, Colpaert CG, Dirix LY, Van Marck EA, Vermeulen PB. Letter to the editor: Lymphangiogenesis in primary breast cancer. Cancer Lett. 2007 Oct 28;256(2):279-81; author reply 283-4.
• Van den Eynden GG, Van der Auwera I, Van Laere SJ, Trinh XB, Colpaert CG, van Dam P, Dirix LY, Vermeulen PB, Van Marck EA. Comparison of molecular determinants of angiogenesis and lymphangiogenesis in lymph node metastases and in primary tumours of patients with breast cancer. J Pathol. 2007 Sep;213(1):56-64.
• Van den Eynden GG, Colpaert CG, Couvelard A, Pezzella F, Dirix LY, Vermeulen PB, Van Marck EA, Hasebe T. A fibrotic focus is a prognostic factor and a surrogate marker for hypoxia and (lymph)angiogenesis in breast cancer: review of the literature and proposal on the criteria of evaluation. Histopathology. 2007 Oct;51(4):440-51.
• Van den Eynden GG, Van Laere SJ, Van der Auwera I, Gilles L, Burn JL, Colpaert C, van Dam P, Van Marck EA, Dirix LY and Vermeulen PB. Differential expression of hypoxia and (lymph)angiogenesis-related genes at different metastatic sites in breast cancer. Clin Exp Metastasis 2007 Feb 13
• Van den Eynden GG, Van der Auwera I, Van Laere SJ, Huygelen V, Colpaert CG, van Dam P, Dirix LY, Vermeulen PB and Van Marck EA. Induction of lymphangiogenesis in and around axillary lymph node metastases of patients with breast cancer. Br J Cancer 2006 Nov 20; 95(10): 1362-6
• Van den Eynden GG, Van der Auwera I, Van Laere SJ, Colpaert CG, van Dam P, Dirix LY, Vermeulen PB, Van Marck EA. Distinguishing blood and lymph vessel invasion in breast cancer: a prospective immunohistochemical study. Br J Cancer. 2006 May 2; [Epub ahead of print]
• Van den Eynden GG, Van der Auwera I, Van Laere SJ, Colpaert CG, Turley H, Harris AL, van Dam P, Dirix LY, Vermeulen PB and Van Marck EA. Angiogenesis and hypoxia in lymph node metastases is predicted by the angiogenesis and hypoxia in the primary tumour in patients with breast cancer. Br J Cancer. 2005 Nov 14;93(10):1128-1136.
• Stessels F, Van den Eynden G, Van der Auwera I, Salgado R, Van den Heuvel E, Harris AL, Jackson DG, Colpaert CG, van Marck EA, Dirix LY, Vermeulen PB. Breast adenocarcinoma liver metastases, in contrast to colorectal cancer liver metastases, display a non-angiogenic growth pattern that preserves the stroma and lacks hypoxia. Br J Cancer. 2004 Apr 5;90(7):1429-36.