Abstract A47: Circulating tumor cell (CTC) assay development for detection of phosphohistone H3 (pHH3) as a clinical pharmacodynamic (PD) marker for LY2523355, an Eg5 kinesin inhibitor.

Abstract

Abstract Circulating tumor cells have been shown to be prognostic of survival in metastatic breast, colon, and prostate cancers. Additionally, CTCs are of interest because they may be representative of the phenotype/genotype of the primary and metastatic tumors, and may be a useful tool (i.e. patient stratification) for drug development. CTCs, as “liquid biopsies” are potentially useful as a PD marker as it allows easy repeat sampling before and after drug treatment. We describe the development of a CTC assay that measures the induction of pHH3 by LY2523355, a selective small molecule inhibitor of the human Eg5 kinesin. pHH3 is a biomarker of mitosis. In tumor xenograft mouse models, LY2523355 treatment was shown to reduce tumor volume with elevation of pHH3 in tumors. The CTC assay was developed using the FDA cleared Veridex/CellSearch™ instrument and reagents for enumeration of CTCs from blood. CellSearch defines CTCs as EpCam+/DAPI+/CK 8, 18, 19+/CD45−. For assay development, cells from tumor cell lines representing major solid tumor types were chosen, cultured, treated with LY2523355, or with various standard-of-care chemotherapeutics, and spiked into whole blood drawn into CellSave™ (preservative+EDTA). 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 CellSearch Mouse/Rat CTC kit (for mouse blood) or human CXC kit (for human blood), supplemented with the R-PE conjugated anti-pHH3 antibody. pHH3 in the recovered tumor cells was detected in the open/fourth channel on the CellSearch instrument. About 1–4% of all cultured tumor cells were positive for pHH3 without drug treatment. Significant induction of pHH3 (30–50% cells) in sensitive tumor cell lines was observed with 24 hour treatment with only drugs interfering with mitosis (paclitaxel, taxotere, and LY2523355). The magnitude of the induction of pHH3 in the cells after treatment was dose and cell-line dependent. A dose response of pHH3 induction was observed with 0.5nM-25nM of LY2523355 with several cultured tumor cell lines (Colo205, HCT116, H441, SKBR3, MDA-MB-468), spiked into blood, and assayed using CellSearch. The concentrations of the dose response are similar to the drug exposure levels of that of preclinical tumor xenograft studies. LY2523355 is currently in early phase clinical development, and monitoring CTCs with pHH3 expression post-treatment may be a useful PD marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A47.