Phosphorylation of histone H2AX (γH2AX) occurs in response to DNA double-strand breaks induced by a variety of genotoxic treatments. The number of resulting microscopic γH2AX foci detected by immunofluorescence has been correlated directly with DNA damage severity and genomic repair. Our goal has been to develop and validate γH2AX immunocytochemical assays to monitor response to DNA damage during Phase 0/1 clinical trials of Topo-1 inhibitors conducted by the National Cancer Institute. Since the Phase 0 trial has no therapeutic benefit to currently enrolled patients, it is critical to minimize the risk with biological active doses of treatment and limited biopsies for measuring pharmacodynamic (PD) biomarker on tumor targets. By reliably monitoring PD response of surrogate specimens such as PBMC from whole blood, we provide a basis for triggering tumor biopsy and examining tumor response. We used the human monocytic leukemia (THP-1) cell line for assay development, mouse treatment trials as pre-clinic models for assay optimization, and human blood ex vivo for clinical trial feasibility and assay validation. Slides were prepared and processed using fluorescent antibody staining for γH2AX foci. We tested various doses of ionizing radiation and DNA topoisomerase I (Topo-1) inhibitors including camptothecins and indenoisoquinoline derivatives by quantifying γH2AX foci after staining with fluorescent conjugated monoclonal antibody. We developed positive and negative controls for the assay, as well as quantitative panels for analysis of γH2AX staining. Multiplexed fluorescent immunoassays were analyzed for biomarker co-localization and cell lineage identification. Assay performance in cancer cell lines, mouse bone marrow PBMCs and splenocytes, as well as human blood ex vivo treatments supports γH2AX in PBMC as a robust pharmacodynamic surrogate biomarker with substantial clinical potential for monitoring tumor response to agents producing DNA damage during early clinical trials. Funded by NCI Contract N01-CO-12400. .
AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics-- Oct 22-26, 2007; San Francisco, CA