Semi-automated immunohistochemical staining of the VEGF-A-protein for clinical use and the identification in NHG-graded breast carcinoma

Abstract: Angiogenesis has a crucial influence on tumour development and identification of microvessels in malignant breast cancer tissue is an indicator for worse prognosis. Angiogenesis is partially governed by the family of vascular endothelial growth factors (VEGF) and their receptors, by which the VEGF-A-protein seems to be the most important factor.     The aims of this work were to first establish a method for immunohistological (IH)-staining of the VEGF-A-protein for clinical use and then to label and evaluate the expression of this protein in 31 Nottingham Histology Graded (NHG I-III) breast carcinoma.      Formaldehyde-fixated tissues from invasive breast neoplasms and control tissues were labelled with monoclonal antibodies against VEGF-A and CD31-proteins using a semi-automated IH-system from Ventana BenchMark. Positively stained vessels were counted from digital copies of microscopic pictures related to mm2 tissue.     A method of IH-labelling with VEGF-A protein was successfully established before staining of the breast tissue and in 19 of the 31 breast cancers. Vessels were counted for both antibodies. The VEGF-A-antibody stained 2.7 ± 2.3 (mean ± SD) vessels/mm2 and the CD31-antibody stained 27.3 ± 19.3 in the breast carcinoma tissue. The percent of VEGF-A-stained vessels in relation to CD31-stained were 7.6% in the NHG-I- (n=3), 7.8% in the NHG-II- (n=10) and 15.0% in the NHG-III-group (n=6).     The results demonstrate that increased NHG-grade and lower differentiation can be associated with higher percent of vessels expressing VEGF-A-protein. The result was not statistically certified because of the small number of stained breast cancers and additional investigations are recommended before clinical use.