Establishment of a highly sensitive Taqman-based assay for non-invasively detecting biomarkers expressed in circulating tumor cells in human and mouse whole blood.

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 3492 In this study, we developed a Taqman-based RT-PCR assay to quantify circulating tumor cells (CTCs) in human and mouse whole blood. Human gastric cancer cells MKN45 were spiked into whole mouse or human blood and RNA was extracted and analyzed by Taqman RT-PCR with probes specific for a panel of markers, including human Cytokeratin 8 (CK-8), Cytokeratin 18 (CK-18), Cytokeratin 19 (CK-19), cMET and EpCAM. The assay can detect as few as 2-5 cells in 1ml of human or mouse whole blood. The sensitivity of the assay depends on the expression level of the marker used, with CK-18 being the most abundant marker for MKN45 and hence the most sensitive one. To correlate tumor burden with amount of human cancer markers in mouse blood in an animal model, MKN45 cells were implanted i.p. to nude mice, and after 3 weeks peritoneal tumors were excised and weighed and blood analyzed. The amount of CTCs, as measured by Taqman on RNA, tightly correlated (R2 = 0.8893) with tumor burden. To evaluate whether such markers can be used as surrogate markers for predicting treatment efficacy, a set of nude mice bearing MKN45 i.p. tumors was treated with 25 or 75 mg/kg/dose p.o. twice daily for 2 weeks with a potent inhibitor of cMet as well as a potent inhibitor CPT-11. The cMET compound caused a 50% and 90% reduction in tumor burden, whereas CPT-11 caused over 90% tumor reduction. The blood was analyzed and showed a correlated dose-dependent reduction in the markers by the Taqman assay. Our data suggest that human cancer/epithelial markers can be used as surrogate markers for assessing efficacy during anti-cancer drug treatment. Additionally, such markers have potential for patient stratification using non-invasive methods. contact: drhanying@gmail.com