Purpose: In a recent study, we presented preliminary evidence for biological activity in a Phase I dose-finding study (15-600 mg/m 2 ) of recombinant human endostatin in patients with refractory solid tumors. Here, we conducted additional biomarker analyses to correlate changes in tumor biology with dose. Experimental Design: Excisional tumor biopsies were obtained at baseline and after 56 days of endostatin therapy. Laser scanning cytometry (LSC) was used to quantify biomarker levels in whole

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... issue sections. Apoptosis in tumor cells (TCs) and tumor-associated endothelial cells (ECs) was quantified by fluorescent three-color anti-CD31/terminal deoxynucleotidyl transferase-mediated nick end labeling staining. Microvessel densities were measured by LSCguided vessel contouring. Levels of tumor-associated EC BCL-2 and hypoxia-inducible factor 1␣ were determined by immunofluorescence and LSC quantification. The results, including tumor blood flow measured by positron emission tomography, were analyzed using a quadratic polynomial model. Results: Significant increases in EC death and decreases in tumor microvessel density were observed, with maximal effects of endostatin at a dose of 249 mg/m 2 (95% confidence interval, 159 -338) and 257 mg/m 2 (95% confidence interval, 183-331), respectively. In contrast, levels of TC death were uniformly low and did not correlate with endostatin dose. Maximal nuclear hypoxia-inducible factor 1␣ and minimal EC Bcl-2 levels were observed at ϳ250 mg/m 2 , although the changes did not reach statistical significance. Conclusions: The data suggest that endostatin had optimal biological activity at doses ϳ250 mg/m 2 in our cohort of patients. Endostatin's failure to induce high levels of TC death may explain its lack of significant clinical activity in this Phase I trial.