NOVEL METHODS FOR THE DETERMINATION OF THE ANGIOGENIC ACTIVITY OF HUMAN TUMORS

At present the most used method to quantify tumor angiogenesis in huma n solid tumors is the count of intratumoral microvessels in the primar y lesion. This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microv essels. Several studies have found that intratumoral microvessel densi ty (IMD) determined in the primary tumor is significantly associated w ith metastasis and prognosis in some solid neoplasia, particularly in operable breast carcinoma. The subjective evaluation of IMD made by tw o observers at the microscope is rapid and of low cost, but presents s ome difficulties, mainly the identification of the most vascularized a rea (''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 syst em (CIAS) seems to be a promising tool to improve accuracy, feasibilit y and reproducibility of microvessel counts, although there are still some open technical problems to completely automate its use. Angiogeni c activity is the result of a balance between angiogenic stimuli and a ngio-inhibition, Therefore the determination of angiogenic peptides an d/or natural angiogenesis inhibitors in the tumor tissue, serum, or ur ine of cancer patients seems to be a promising alternative to microves sel counting. At present it is possible to determine the expression of basic fibroblast growth factor (bFGF), vascular endothelial growth fa ctor, and transforming growth factor beta using immunohistochemical me thods. Serum and urine levels of bFGF can be assessed using an immunoe nzymatic assay. Methods used to assess the expression and levels of ur okinase-type plasminogen activator (uPA) or plasminogen activator inhi bitor-1 (PAI-1) have also been developed, and correlate with angiogeni c 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 seem s 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 t umor. For a reliable and reproducible assessment of angiogenesis for a ll of the assays, validation procedures and quality control protocols are mandatory.