Article

Development of a Validated Immunofluorescence Assay for H2AX as a Pharmacodynamic Marker of Topoisomerase I Inhibitor Activity

Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Support Directorate, Science Applications International Corporation-Frederick, Inc., NationalCancer Institute-Frederick, Frederick, MD 21702, USA.
Clinical Cancer Research (Impact Factor: 8.19). 10/2010; 16(22):5447-57. DOI: 10.1158/1078-0432.CCR-09-3076
Source: PubMed

ABSTRACT Phosphorylated histone H2AX (γH2AX) serves as a biomarker for formation of DNA double-strand break repair complexes. A quantitative pharmacodynamic immunofluorescence assay for γH2AX was developed, validated, and tested in human tumor xenograft models with the use of clinically relevant procedures.
The γH2AX immunofluorescence assay uses a novel data quantitation and image processing algorithm to determine the extent of nuclear-specific γH2AX staining in tumor needle biopsies and hair follicles collected from mice bearing topotecan-responsive A375 xenografts. After method validation with the topoisomerase I (Top1) inhibitor topotecan, the assay was used to compare pharmacodynamic properties of three structurally related indenoisoquinoline Top1 inhibitors.
γH2AX response to topotecan was quantified over a 60-fold dose range (0.016-1.0 times the murine single-dose maximum tolerated dose), and significant pharmacodynamic response was measured at the mouse equivalent of the 1.5 mg/m(2) clinical dose as well as the lowest dose tested. Responses were within a time window amenable for biopsy collection in clinical trials. These studies enabled characterization of dose and time responses for three indenoisoquinolines, resulting in selection of two for clinical evaluation. γH2AX response to Top1 inhibitors in hair follicles was also observable above a minimal dose threshold.
Our γH2AX assay is sufficiently accurate and sensitive to quantify γH2AX in tumor samples and will be used in correlative studies of two indenoisoquinolines in a phase I clinical trial at the National Cancer Institute. Data suggest that hair follicles may potentially serve as a surrogate tissue to evaluate tumor γH2AX response to Top1 inhibitors.

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    • "Skin biopsies are necessary to obtain the basal keratinocytes, an issue which limits their routine use due to its invasive nature. It is noticeable in sections of skin biopsies that hair follicle cells often exhibit the largest γ-H2AX response after drug treatment [56] [57]. An alternative, and less invasive procedure for obtaining at least some of these follicle cells, is plucking [58] [59]. "
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