CCR Translations Bringing DNA Repair in Tumors into Focus

In this issue of Clinical Cancer Research, Bañuelos and colleagues assess H2AX phosphorylation as a predictive biomarker of response to DNA damaging agents that cause double strand breaks (DSBs) (ref. 1). DSBs, where breaks in both strands of DNA occur in close vicinity, are especially lethal to replicating cells. This sensitivity has long been exploited in cancer therapy; radiotherapy and a wide range of chemotherapies (e.g., cisplatin, temozolomide, camptothecin, etoposide, cytarabine, gemcitabine etc.) all cause DSB formation. This occurs either directly, as is the case with ionizing radiation (IR) and bleomycin, or by indirect routes such as replication fork stalling, an event that can lead to fork collapse and DSB formation. Even some of the novel highly selective/less toxic classes of anticancer therapies, such as the PARP inhibitors (e.g., Olaparib) rely, in part, on the tried and tested approach of causing DSB formation (2). Despite the widespread use of these therapies, robust biomarkers of DSB formation that could be used to predict and monitor drug efficacy and response are not available. Moreover, intertumoral and interindividual variation in these responses might explain the variability in the clinical efficacy of therapies. One potential biomarker of DSB formation and repair is phosphorylation of the histone H2AX. H2AX is a member of the histone H2A family, one of the five histone types that package DNA into chromatin. Each nucleosome, the basic subunit of chromatin, contains two H2A molecules, of which approximately 10% are H2AX. Shortly after DSB formation, PI3-family kinases such as ATM, ATR, and DNAPK phosphorylate a number of proteins, including H2AX. Via a series of protein-protein interactions, the phosphorylated form of H2AX, γH2AX, initiates DNA repair processes and the stalling of the cell cycle while DNA is repaired (3). In cells in culture, γH2AX can be detected by immunofluorescence as distinct nuclear foci, as early as 30 minutes after damage. As DSBs are repaired, the number of γH2AX foci present per nucleus gradually decreases and thus the elevation and decline of γH2AX foci reflects the activation of the DSB repair apparatus, as well as the eventual repair of DSBs (Fig. 1). Because of the role of H2AX and the ability to detect γH2AX by approaches such as flow cytometry (FACS) or immunohistochemistry, Bañuelos and colleagues and others have previously investigated the potential of γH2AX as a biomarker. For example, γH2AX levels can predict the response to chemotherapeutics in vitro (4, 5) and pilot studies have assessed the potential of γH2AX as a nontumor tissue pharmacodynamic (PD) marker of DNA damage in radiotherapy and PARP inhibitor trials (3, 6). γH2AX has also been assessed as a diagnostic biomarker, both in rectal cell carcinoma and melanocytic lesions (7, 8). In this new article (1), Bañuelos and colleagues extend their studies to assess the potential of γH2AX as a predictive biomarker of tumor response to radioor chemotherapy in vivo. As an experimental model, Bañuelos and colleagues used human tumor cell lines xenografted into mice that were subsequently treated with either cisplatin, IR, or a cisplatin-IR combination mirroring a clinical protocol used for advanced cervical cancer. To assess predictive power, γH2AX foci from treated xenografts were quantified and compared to the in vitro clonogenic survival of cells derived from the same xenografts. As in previous studies by these authors (9), a reduced γH2AX response 24 hours after treatment predicted higher survival rates of tumor cells in vitro, especially when γH2AX was assessed by immunohistochemistry, rather than FACS. The greater power of immunohistochemical detection perhaps reflects the consensus opinion that γH2AX foci most likely represent the response to DSB formation, whereas the total amount of γH2AX, as detected by FACS, also includes phosphorylation not foci related nor necessarily caused by DSB formation (3). The correlations between γH2AX focus formation in xenografts and the in vitro clonogenic survival of cells are persuasive but it is critical that this biomarker is assessed in patient samples. To achieve this, Bañuelos and colleagues examinedγH2AX foci in tumor material from cervical cancer patients treated with cisplatin and IR. Importantly, the authors show that with the correct sample preparation procedure, it is feasible to measure γH2AX foci in the formalin-fixed-paraffin-embedded biopsies that are commonly used in clinical pathology. By examining biopsies both before and after treatment, they also provide vital proofof-principal data suggesting that treatment-induced increases in γH2AX can be detected in biopsy material. Despite the small Authors' Affiliation: The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, United Kingdom Received 2/24/09; accepted 2/27/09; published online 5/15/09. Requests for reprints: Christopher J. Lord and Alan Ashworth, Institute of Cancer Research, Chester Beatty Labs, 237 Fulham Road, London, SW3 6JB, United Kingdom. Phone: 44-20-7153-5333; Fax: 44-20-7153-5340; E-mail: [email protected] or [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-09-0434