Expression profiling of blood samples from an SU5416 Phase III metastatic colorectal cancer clinical trial: a novel strategy for biomarker identification

BACKGROUND: Microarray-based gene expression profiling is a powerful approach for the identification of molecular biomarkers of disease, particularly in human cancers. Utility of this approach to measure responses to therapy is less well established, in part due to challenges in obtaining serial biopsies. Identification of suitable surrogate tissues will help minimize limitations imposed by those challenges. This study describes an approach used to identify gene expression changes that might serve as surrogate biomarkers of drug activity. METHODS: Expression profiling using microarrays was applied to peripheral blood mononuclear cell (PBMC) samples obtained from patients with advanced colorectal cancer participating in a Phase III clinical trial. The PBMC samples were harvested pre-treatment and at the end of the first 6-week cycle from patients receiving standard of care chemotherapy or standard of care plus SU5416, a vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitor. Results from matched pairs of PBMC samples from 23 patients were queried for expression changes that consistently correlated with SU5416 administration. RESULTS: Thirteen transcripts met this selection criterion; six were further tested by quantitative RT-PCR analysis of 62 additional samples from this trial and a second SU5416 Phase III trial of similar design. This method confirmed four of these transcripts (CD24, lactoferrin, lipocalin 2, and MMP-9) as potential biomarkers of drug treatment. Discriminant analysis showed that expression profiles of these 4 transcripts could be used to classify patients by treatment arm in a predictive fashion. CONCLUSIONS: These results establish a foundation for the further exploration of peripheral blood cells as a surrogate system for biomarker analyses in clinical oncology studies

The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts

SU5416 and SU6668 are potent antiangiogenic small-molecule inhibitors of receptor tyrosine kinases, including those of the vascular endothelial growth factor and platelet-derived growth factor receptor families. The stem cell factor (SCF) receptor, c-kit, is structurally related to these receptors and, although not expressed on mature peripheral blood cells, is expressed in leukemic blasts derived from 60% to 80% of acute myeloid leukemia (AML) patients. The c-kit kinase inhibitory activity of SU5416 and SU6668 was evaluated in MO7E cells, a human myeloid leukemia cell line. Tyrosine autophosphorylation of the receptor, induced by SCF, was inhibited in these cells by SU5416 and SU6668 in a dose-dependent manner (inhibitory concentration of 50% [IC50] 0.1-1 μM). Inhibition of extracellular signal–regulated kinase 1/2 (ERK1/2) phosphorylation, a signaling event downstream of c-kit activation, was also inhibited in a dose-dependent manner. Both compounds also inhibited SCF-induced proliferation of MO7E cells (IC50 0.1 μM for SU5416; 0.29 μM for SU6668). Furthermore, both SU5416 and SU6668 induced apoptosis in a dose- and time-dependent manner as measured by the increase in activated caspase-3 and the enhanced cleavage of its substrate poly(ADP-ribose) polymerase. These findings with MO7E cells were extended to leukemic blasts from c-kit+ patients. In patient blasts, both SU5416 and SU6668 inhibited SCF-induced phosphorylation of c-kit and ERK1/2 and induced apoptosis. These studies indicate that SU5416 and SU6668 inhibit biologic functions of c-kit in addition to exhibiting antiangiogenic properties and suggest that the combination of these activities may provide a novel therapeutic approach for the treatment of AML.</jats:p