Sensitive detection of gamma-H2AX induction as a pharmacodynamic marker for profiling patients with cancer treated with topotecan.

Abstract

3093 Background: Chemotherapies for cancer patients are often designed to inhibit growth or induce apoptosis in malignant cells. Several genotoxic agents have proved effective in inducing significant DNA damage, such that apoptosis is triggered in the cell. Topotecan is a chemical inhibitor of DNA topisomerases. The inhibition of topisomerases blocks the ligation of the phosphate backbone of DNA and leads to the generation of strand breaks. In this study we evaluated gamma-H2AX as marker for drug efficacy, using antibodies that recognize the phosphorylated S139 epitope. Results: We present an automated blood-based assay for detecting phosphorylated gamma-H2AX before and after drug treatment. We first screened subpopulations of the blood for cell types that exhibit the great response in gamma-H2AX induction after drug treatment. Using this sub-phenotype, we then developed two approaches for isolating these cell types: immunomagentic enrichment of the top responding cell types, and immunophenotyping of the top responding cell subtypes. We measured gamma-H2AX, using laser scanning cytometry and found that the induction magnitude of the biomarker (1) correlates to treatment with the topotecan in set of ex vivo patient samples and in clinical trial patients; and (2) and is at least as sensitive as manually measurements of gamma-H2AX foci using conventional methods in comparison of 10 patient samples. We have begun a clinical validation study to determine whether this induction correlates to clinical outcome and additionally are investigating whether early response to topotecan, in the form of biomarker induction, is predictive of outcome. Preliminary results in 10 patient samples show that the early gamma-H2AX response to DNA damage is highly variable from patient to patient, ranging from no measurable induction, to an induction of 65%. Conclusions: We have developed an automated system for measuring gamma-H2AX induction in cancer patients treated with topotecan. Studies correlating the marker to outcome are ongoing. If this assay is predictive, it would be invaluable for rapidly stratifying patients in clinical trials that involve therapies that induce DNA damage. No significant financial relationships to disclose.