REVIEWS Drug Discovery Today  Volume 12, Numbers 19/20  October 2007 Molecular and cellular biomarkers for angiogenesis in clinical oncology Francesco Bertolini 1 , Patrizia Mancuso 1 , Yuval Shaked 2 and Robert S. Kerbel 2 1 Division of Hematology-Oncology, Department of Medicine, European Institute of Oncology, 20141 Milan, Italy 2 The Sunnybrook Health Sciences Centre, Molecular and Cellular Biology, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M4N 3M5, Canada Medical oncologists are increasingly using anti-angiogenic drugs, but identifying the best-suited drug and the optimal dosage and schedule for treatment of patients remain challenging issues. Circulating endothelial cells (CECs) and circulating endothelial progenitors (CEPs) are modulated in a variety of diseases including cancer, and are promising surrogate biomarkers in oncology. Molecular surrogate markers, on the other hand, are more scanty at the present time, because the identification of truly endothelial cell-restricted genes and/or antigens is complex. Here, we discuss the biological and technical facets of the search and validation of new biomarkers for angiogenesis. A number of ‘molecularly targeted’ anti-angiogenic drugs such as bevacizumab (Avastin 1 ), ranibizumab (Lucentis), sunitinib (Sutent 1 ), and sorafenib (Nexavar) are now available for clinical oncologists and ophtalmologists [1–4]. Their clinical use, however, suffers from several relevant limitations. Although some large clinical trials have demonstrated a benefit of these drugs in terms of prolonged survival of cancer patients or increased visual func- tion in patients with blinding conditions such as age-related macular degeneration (AMD), there is a compelling need for determining the optimal biologic dose (OBD) of these drugs, monitoring their biologic activity, selecting and stratifying the patients who are most likely to benefit from treatment. In medical oncology, problems related to the definition of the OBD for such drugs include the low frequency of tumor responses (tumor shrink- age); the lack, in some cases, of dose limiting toxicities (DLT) normally used to define a maximum tolerated dose (MTD), observed frequently when using cytotoxics but not as frequently when using certain anti-angiogenic drugs; and (in either oncology and ophthalmology) significant (if not optimal) therapeutic activ- ity at doses below the MTD. Most of these drugs are extremely expensive [5,6], and the escalating cost of clinical care underlines the urgent need for development and clinical validation of bio- markers of angiogenesis for patient selection and stratification and for OBD tailoring [1–4,7]. Molecular and imaging strategies to measure angiogenesis and anti-angiogenic drug activity A number of preclinical angiogenesis assays and models have been generated. Growth factor (VEGF or FGF)-induced generation and quantification of new vessels is possible in the cornea or in the skin of some animal models, for example mice or rats [4]. The chick embryo chorioallantoic membrane (CAM) assay is based on the implantation of a gelatin sponge with the agent on the top of the growing CAM on day 8 of development. In this technique, after implantation, the sponge is treated with a stimulator of blood vessel formation in the absence or presence of an angiogenesis inhibitor, and evaluated for neovessel formation [8]. These pre- clinical approaches have limitations: some are poorly standar- dized, some are difficult to reproduce and in many cases reference values are scanty. Moreover, these invasive measure- ments are not adaptable to patients. Thus, the evaluation of the efficacy of a given anti-angiogenic therapy so far has been mainly based on the measurement of microvessel density (MVD) in biopsy samples [9]. In this approach, blood vessels are visualized by immunohistochemistry for endothelial-associated antigens, for example CD31, CD34, CD105 or von Willebrand factor, vWf. Positive vessels are counted under the microscope in a manual Reviews  GENE TO SCREEN Corresponding author: Bertolini, F. (francesco.bertolini@ieo.it) 806 www.drugdiscoverytoday.com 1359-6446/06/$ - see front matter ß 2007 Published by Elsevier Ltd. doi:10.1016/j.drudis.2007.08.011