Abstract 5437: A novel method to quantify gamma H2AX foci in circulating tumor cells in patients receiving chemotherapy for colorectal cancer
Authors: Saggese Matilde , Ensell Leah , Vesely Clare , Spanswick Victoria , Peruzzi Elena , Cunati Diana , Signorini Giulio , Hartley John , Arkenau Hendrik Tobias , Meyer Tim 
Published: August 2015

Abstract
Phosphorylated H2AX (γH2AX) is a marker of DNA double-strand break damage and has been proposed as a pharmacodynamic biomarker following treatment with chemotherapy. γH2AX levels in circulating tumor cells (CTCs) may provide a sensitive direct marker for assessing drug effects in tumor tissue without the need for biopsy. In the present study, we established a novel assay to quantify changes in γH2AX in CTCs from patients with metastatic colorectal cancers undergoing treatment with Fulfox or Folfiri. γH2AX foci were initially assessed in human colon adenocarcinoma cancer cell lines (HT-29) treated with different concentrations of Oxaliplatin and SN-38 at different time points. Image capture was conducted using Confocal Microscope Leica SPE2 and γH2AX foci were analyzed with CellProfiler software. The highest number of γH2AX foci/nucleus was induced at 5uM for Oxaliplatin and 0.01uM for SN-38 and the peak of γH2AX foci/nucleus was observed between two-six hours post dose. To reproduce the analytical process for CTCs, we evaluated the γH2AX signal using both the CellSearch System (Janssen Diagnostics) and the DEPArray™ System (Silicon Biosystems). HT-29 cells were used, either as untreated controls or following 2 hours treatment with Oxaliplatin 5uM or SN-38 0.01uM and spiked in to healthy donor blood. Cells were defined as positive for γH2AX on the CellSearch according to a previously validated assay. For Oxaliplatin and SN-38 treated cells, 15.28% and 18.37% respectively were classified as positive for γH2AX, compared with 5.10% for untreated controls. However, using CellSearch, the fluorescent signal could not be quantified and we therefore repeated the experiment using the DEPArray platform. HT-29 cells were treated with SN-38 as above, and compared with an untreated control group using two different exposure times for fluorescein isothiocyanate-conjugated antibody (FITC): FITCI (100 ms and gain 1X) and FITCII (800 ms and gain 4X). The treated group showed a significantly increased intensity of FITC staining compared with the untreated control group: mean 363 vs mean 220 (p&lt;0.0001) for FITCI, and mean 5521 vs mean 4365 (p&lt;0.0040) for FITCII. The DEPArray system therefore has an advantage in being able to quantify differences in signal intensity caused by induction of γH2AX in CTCs and then select individual cells for further downstream analysis. This analysis is now being applied to patient samples collected pre and post-chemotherapy to evaluate the utility of the assay in a clinical setting.
Citation Format: Matilde Saggese, Leah Ensell, Clare Vesely, Victoria Spanswick, Elena Peruzzi, Diana Cunati, Giulio Signorini, John Hartley, Hendrik Tobias Arkenau, Tim Meyer. A novel method to quantify gamma H2AX foci in circulating tumor cells in patients receiving chemotherapy for colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5437. doi:10.1158/1538-7445.AM2015-5437