At present the most used method to quantify tumor angiogenesis in human solid tumors is the count of intratumoral microvessels in the primary lesion. This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microvessels. Several studies have found that intratumoral microvessel density (IMD) determined in the primary tumor is significantly associated with metastasis and prognosis in some solid neoplasia, particularly in operable breast carcinoma. The subjective evaluation of IMD made by two observers at the microscope is rapid and of low cost, but presents some difficulties, mainly the identification of the most vascularized area (''hot-spot'') within each tumor. This method can be improved upon to attain a better reproducibility among different pathologists. For example, the use of a multiparametric computerized image analysis system (CIAS) seems to be a promising tool to improve accuracy, feasibility and reproducibility of microvessel counts, although there are still some open technical problems to completely automate its use. Angiogenic activity is the result of a balance between angiogenic stimuli and angio-inhibition, Therefore the determination of angiogenic peptides and/or natural angiogenesis inhibitors in the tumor tissue, serum, or urine of cancer patients seems to be a promising alternative to microvessel counting. At present it is possible to determine the expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and transforming growth factor beta using immunohistochemical methods. Serum and urine levels of bFGF can be assessed using an immunoenzymatic assay. Methods used to assess the expression and levels of urokinase-type plasminogen activator (uPA) or plasminogen activator inhibitor-1 (PAI-1) have also been developed, and correlate with angiogenic activity and prognosis of patients with breast cancer. Finally, some investigational methods to assess angiogenesis in vivo are presented and discussed. Angiogenesis is a very complex phenomenon. Thus it seems reasonable to hypothesize that its assessment by using concurrently several of the available methods may provide more valid, accurate, and comprehensive information on the angiogenic activity of each single tumor. For a reliable and reproducible assessment of angiogenesis for all of the assays, validation procedures and quality control protocols are mandatory.
