Development of a Validated Immunofluorescence Assay for gamma 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: International efforts to sequence the genomes of various human cancers have been broadly deployed in drug discovery programmes. Diagnostic tests that predict the value of the molecularly targeted anticancer agents used in such programmes are conceived and validated in parallel with new small-molecule treatments and immunotherapies. This approach has been aided by better preclinical cancer models; an enhanced appreciation of the complex interactions that exist between tumour cells and their microenvironment; the elucidation of interactions between many of the genetic drivers of cancer, including oncogenes and tumour suppressors; and recent insights into the genetic heterogeneity of human tumours made possible by extraordinary improvements in DNA-sequencing techniques. These advances are being employed in the first generation of genomic clinical trials that will examine the feasibility of matching a broad range of systemic therapies to specific molecular tumour characteristics. More-extensive molecular characterization of tumours and their supporting matrices are anticipated to become standard aspects of oncological practice, permitting continuous molecular re-evaluations of human malignancies on a patient-by-patient and treatment-by-treatment basis. We review selected developments in translational cancer biology, diagnostics, and therapeutics that have occurred over the past decade and offer our thoughts on future prospects for the next few years.
    Nature Reviews Clinical Oncology 10/2014; 11(11). DOI:10.1038/nrclinonc.2014.158 · 15.70 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Aim Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. Methods We tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. Results In a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. Conclusion Taken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development.
    European Journal of Cancer 10/2014; 50(15):2725-2734. DOI:10.1016/j.ejca.2014.07.004 · 4.82 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Camptothecin and its derivatives, topotecan and irinotecan are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821, confirmed marked antiproliferative synergy with camptothecin, and even greater synergy with LMP-400. Single cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970 enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof-of-principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.
    Cancer Research 09/2014; 74(23). DOI:10.1158/0008-5472.CAN-13-3369 · 9.28 Impact Factor

Full-text (2 Sources)

Available from
May 31, 2014