Assay Development For Detecting Cmet Expression In Circulating Tumor Cells (Ctc), A Potential Patient Tailoring Marker For Evaluation Of Cmet Inhibitors

Abstract Hepatocyte growth factor and its receptor c-MET have been implicated in tumor formation and progression as well as drug treatment resistance to targeted agents such as EGFR inhibitors. cMET over-expression or gene amplification has been reported in a wide variety of human malignancies correlating with poor patient prognosis. A reasonable hypothesis for experimental agents targeting the cMET receptor is that patients with tumors expressing high levels of cMET may respond better. CTC counts are prognostic of survival in metastatic breast, colorectal, and prostate cancers (NEJM 2004;351:781-91; J Clin Oncol 2008;26:3213-21; Clin Can Res 2008;14:6302-9). CTCs are shed from tumors and are believed to be representative of cells migrating to form distal metastasis. The presence of these cells in blood provides an attractive and easy source for serial monitoring of tumors without the limitations of traditional solid tumor biopsy. We describe here the development of an assay that measures cMET expression in CTCs. The CTC assay was developed using the Veridex CellSearch® System, and RUO reagents for enumeration of CTCs and a Lilly proprietary cMET antibody (Ab) optD11. For assay development, cultured tumor cell lines with differing cMET expression levels representing solid epithelial tumor types were spiked into whole blood drawn into a CellSave tube. For initial assay development, mouse whole blood was used and results reproduced subsequently with human whole blood from healthy subjects. The spiked tumor cells in blood samples were recovered using the CellCapture™ Mouse/Rat CTC kit (for mouse blood) or CellSearch® CXC kit (for human blood), supplemented with the fluorescent dye (R- phycoerythrin) conjugated anti-cMET Ab. cMET in the recovered tumor cells was detected in the open/fourth channel on the CellTrack® Analyzer II®. Several proprietary and commercial cMET antibodies were screened for specificity and sensitivity. A commonly used commercially available cMET Ab (Santa Cruz Biotechnology C28) was not able to discriminate between cMET negative and positive cell lines. Results were confirmed by flow cytometry and by western blot analyses. optD11 was selected for CTC cMET expression assay development for the following reasons: a) ability to detect different cMET levels among positive cell lines (H441, MKN45, SNU5, SKOV3) while identifying SKBR3 as cMET negative; b) high affinity for cMET to allow for an image acquisition time of 0.02-0.04 second in the open channel; c) tolerance to the presence of LY2875358 (LA480), an experimental therapeutic anti-cMET Ab, allowing evaluation of cMET levels in CTCs post-treatment. The CTC assay for cMET expression, as well as a CTC assay measuring cMET gene amplification are currently being implemented in the early phase clinical studies for Lilly cMET inhibitors (JSBA, NCT01285037 & JTBA, NCT01287546). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1738. doi:1538-7445.AM2012-1738