Circulating VEGFR2+pAkt+ cells as a functional pharmacodynamic marker in metastatic colorectal cancer treated with antiangiogenic agent

Abstract

Angiogenesis is essential for cancer growth and metastasis. VEGF is a key modulator of angiogenesis and its overexpression is correlated with advanced disease and poor prognosis. Bevacizumab, a recombinant humanized anti-VEGF monoclonal antibody, is the most clinically advanced anti-angiogenic agent. Bevacizumab has received most attention for first-line treatment of advanced colorectal and non-small cell lung cancer and rapid growing evidence for its efficacy in treatment of a number of other solid tumors. However, we are not yet able to sufficiently monitor the activity of this drug. Therefore, effective pharmacodynamic (PD) marker studies to identify those patients responding to bevacizumab or to validate the supposed biological effect, and define the optimum biological dose and schedule are needed. Using an in vitro MTT model, we demonstrated inhibition of human umbilical endothelial cells (HUVECs) proliferation that was associated with reduced VEGFsignaling when HUVECs were treated with bevacizumab compared to cells that were treated with PBS. HUVEC showed low levels of pAkt and pERK in serumfree conditions when examined by Western blotting. However, the addition of VEGF to the medium for 15 min resulted in the phosphorylation of VEGFR-2, Akt and ERK. Bevacizumab was able to inhibit the VEGF-induced autophosphorylation of VEGFR-2, Akt and ERK at 100 ng/ml. Treatment of bevacizumab to DLD-1 tumor-bearing mice showed inhibition of tumor growth compared to tumor treated with PBS. Analysis of VEGFR2 signaling in HUVECs using phospho flowcytometry assay demonstrated that Akt and ERK phosphorylation were induced with treatment of VEGF, while level of VEGFR2 decreased similar to the pattern of Western blot. Adding bevacizumab before stimulation prevented induction of the pAkt and pERK. This techniques and assays showed novel possibilities of PD marker for antiangiogenic agentsThe number of circulating VEGFR2+pAkt+ and VEGFR2+pERK+ cells in the peripheral mononuclear cells in 7 healthy volunteers and 24 patients with mCRC were determined by phospho-flow cytometry. Median percentage of VEGFR2+pAkt+ and VEGFR2+pERK+ cells in cancer patients were 23.44% and 0.35%, increased by 3-fold in both cells compared with healthy volunteer (P< 0.001 and P<0.004 vs control). Bevacizumab treatment resulted in rapid decrease in VEGFR2+pAkt+ cells for 18 out of 24 patients on days 3. The amount ofVEGFR2+pAkt+ cells were increased and normalized on days 15 compared to values on days 3. A total of 17 (70.8%) patients were diagnosed with  1 HTN (grade 1 (n=11), grade 2 (n=4), grade 3 (n=2)), whereas the rest of the patients stayed normotensive (grade 0 (n=7)) on days 3 during bevacizuamb treatment. Bevacizumab combined with chemotherapy decreased VEGFR2+pAkt+ cells due to impaired VEGFR2 signaling in circulating VEGFR2+ cells. Changes in number of VEGFR2+pAkt+ cells level appear to be a promising functional pharmacodynamic marker for drug activity, determination of dosage and administration schedule in the treatment of antiangiogenic agents.