Measurement of gamma-H2AX in circulating tumor cells (CTC) from patients receiving ABT-888 (veliparib) in combination with carboplatin on a phase I dose-escalation study (OSU-10080/NCI8609).

Abstract

58 Background: Non-invasive methods to accurately measure DNA damage induced by PARP inhibitors in the tumors are crucial in identifying the optimal biological dose and schedule of these novel agents. With the use of an antibody that targets the histone H2AX, which becomes phosphorylated on serine residue 139 (γ-H2AX), it is possible to measure DNA DSB in the cell. But repeated biopsy in patients with metastatic breast cancer is not feasible. We propose here to quantify γ-H2AX in CTCs as a measurement of DNA damage in the tumor in patients receiving veliparib (ABT-888), an oral PARP inhibitor and carboplatin on a phase I study. Methods: CTCs were isolated in 10 mL of peripheral blood using negative selection with immunomagnetic tagging and removal of CD45 positive cells at 3 time points. Immunocytochemical staining for nucleus (DAPI), pan-cytokeratin CD45, and γ-H2AX was completed on available samples. Double staining for nucleus and cytokeratin with high nucleus to cytoplasm ratio defined a CTC. The γ-H2AX foci present in the nucleus of the CTCs will be counted by immunocytochemistry (ICC) analysis. Results: We negatively enriched blood sample from enrolled patients with metastatic breast cancer (n=6). CTCs were observed in all samples (55CTCs/mL of blood - 2000CTCs/mL of blood). Thus far 3 out of 6 patient samples have been analyzed. The number of CTCs per mL of blood after enrichment at baseline has a mean of 293, a median of 140 and a range of (684-140) At time point 1 the mean is 1071.33, the median is 1151 and the range is (1151-63). ICC analysis on the CTCs demonstrated the presence of γ-H2AX positive cells. Conclusions: Our novel negative depletion method for isolating CTCs allows for separation of a wider range of CTCs, since it does not assume presence of epithelial markers before separation. This leads to improved yield and purity allowing better ICC detection of various markers directly on CTCs. Development and validation of the above methodology to determine γ-H2AX foci in CTCs will help in identifying a novel, non-invasive biomarker that can be used to assess the effect of DNA damaging agents during therapy.