Abstract A49: Circulating tumor cell (CTC) assay development for detection of H2AX as a clinical pharmacodynamic (PD) marker for Chk1 kinase inhibitors.
Authors: Peek Victoria L. , Um Suzane L. , Yan S. Betty , Yan Lei , Dempsey Jack , Barnard Darlene , King Connie , Ballard Darryl , Heinz-Taheny Kathleen , Marshall Mark , Beckmann Richard , Bence Aimee , Westin Eric H. 
Published: June 2012

Circulating tumor cells are 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 a useful tool (e.g. patient stratification) for drug development. CTCs, as “liquid biopsies” are potentially useful as a pharmacodynamic marker allowing easy repeat sampling before and after drug treatment. We describe the development of a CTC assay measuring H2AX induction, a marker of DNA damage. 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 inhibitors of checkpoint kinase 1 (Chk1, LY2603618, LY2606368) or with various standard-of-care (SOC) chemotherapeutics. These treated cells were then spiked into whole blood drawn into CellSave™ (preservative+EDTA). LY2603618 and LY2606368 are inhibitors of Chk1 kinase currently in clinical development. LY2603618 is meant to be used in combination with a DNA damaging agent, while LY2606368 has potent single agent activity. Inhibition of Chk1 in cells with DNA damage allows the cells to progress into mitosis without DNA repair, leading to cell death. In tumor xenograft mouse models, treatment reduced tumor volume with elevation of γH2AX in tumors and anagen hair follicles at drug exposure levels similar to concentrations used for the CTC γH2AX development assay. 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-γH2AX antibody. γH2AX in the recovered tumor cells was detected in the open/fourth channel on the CellSearch instrument. Results were confirmed by flow analysis. Without drug treatment, only about 0.2–2% of all cultured tumor cells were γH2AX positive. The magnitude of the induction of γH2AX in the cells after treatment was dose and cell-line dependent. Significant induction of H2AX (&gt;20% cells) was observed after 24 hour treatment with only a few SOC agents (cisplatin, etoposide) in sensitive tumor cell lines; the majority induced moderate to low levels (usually &lt;10%) of γH2AX expression at the concentrations tested (gemcitabine, pemetrexed, taxotere, paclitaxel, doxorubicin, bleomycin). In contrast, LY2606368 alone induced γH2AX strongly across a wide variety of cell lines (Calu6, SKOV3, HT29, L3.7, SNU5, H441, DU145, SKBR3). The combination of both Chk1 inhibitors with gemcitabine induced γH2AX to a greater extent than either agent alone. In conclusion, monitoring CTCs with γH2AX expression may be a useful PD marker for clinical studies.

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 A49.