We developed an immunofluorescence-based assay to measure γH2AX response after Top1 inhibitor treatment. The assay uses control and calibrator tissues and was tested using clinically relevant needle biopsy collection and handling procedures (4
). The background fluorescence–induced variability and non-uniformity of nuclear volume within tissue sections, common problems in many assays measuring immunofluorescence, were mitigated with a novel data quantitation and image processing algorithm. Evaluation parameters included specificity of response, amplitude of response measured as fraction of tumor cell nuclei or hair follicle cells positive, timing of response after treatment, and correlation of biomarker response to the biologically effective dose of the Top1 inhibitor topotecan and NSC 724998. After establishing that all of these marker characteristics were consistent with the expected effects of topotecan, we validated the assay for detection of double-strand breaks caused by topotecan administered over a 60-fold dose range (0.016–1.0 MTD) in both topotecan-sensitive and -insensitive human cancer cell-line xenografts and in normal murine testes and small intestine. The assay was then used to measure γH2AX response to three structurally related indenoisoquinoline inhibitors of Top1, NSCs 724998, 725776, and 706774.
It has previously been shown that indenoisoquinolines have activity in camptothecin-resistant cell lines, are chemically more stable than camptothecin, overcome drug efflux-related multidrug-resistance, and produce DNA breaks resistant to reversal of the trapped DNA-Top1 cleavage complex (24
). Despite considerable intra-group heterogeneity in γH2AX response in A375 xenografts after indenoisoquinoline treatment, a response that was significantly different statistically from vehicle was detected for NSCs 724998 and 725776. Notably, γH2AX response was significant compared with vehicle at all doses of NSCs 724998 tested at 4 hours post-dose. This response persisted at 7 hours post-dose. These findings, in conjunction with additional preclinical data, supported selection of NSCs 724998 and 725776 for clinical evaluation (25
). The observation that a significant γH2AX response compared with vehicle could readily be measured in xenografts by 4 hours after drug administration also informed the design of the phase I indenoisoquinolines trial being conducted at the NCI.
It is well established that γH2AX formation occurs in response to DNA double-strand breaks, and apoptosis generates double-strand breaks; the γH2AX assay outlined in this paper cannot separate these two processes. The literature on γH2AX as a marker for DNA double-strand breaks induced by ionizing radiation is extensive; however, less is known about the utility of γH2AX as a marker for monitoring chemotherapy-induced DNA damage. When Rogakou and colleagues used ionizing radiation as a source of double-strand break damage and apoptosis, maximum γH2AX response was estimated to be 9 to 30 minutes post-exposure (10
), which is significantly less than the time course observed in our studies. In contrast, the same group reported that γH2AX started to accumulate 1.5 hours after anti-Fas and TRAIL were used to induce apoptosis in vitro
, corresponding to the appearance of DNA double-strand breaks (34
). Our interpretation of the difference in γH2AX response between our observed drug response and previously published radiation-treated animal models is the difference in the timing of DNA damage. Ionizing radiation results in immediate double-strand break damage. For chemotherapeutic agents, injected as well as oral, there is a time lag between their administration and arrival at the tissue being sampled. In addition, for those agents that act by interfering with DNA metabolism, time is needed for the inhibition and subsequent repair to occur, with most of the double-strand breaks forming during the attempted repair of the breaks themselves. Our ability to determine drug dose response within groups with statistical significance required collecting and testing tumor biopsies no sooner than 4 hours post-dose, which would appear to be a very early time point for detecting apoptosis considering the biodistribution of the compound that must take place in the test animal.
Our use of γH2AX as a pharmacodynamic biomarker also raises the question of quantifying the number of DNA double-strand break repair complexes and using that information to predict cell death in response to drug treatment. It has been reported that cancer cell lines have a wide variation in the number of γH2AX foci per nucleus in the absence of radiation or drug treatment, and these cells continue to grow and divide (36
). The question of whether an increase in the fraction of γH2AX-positive cells reflects an increase in cell death remains to be further explored (35
), although data presented here point in that direction (). We are currently validating an apoptosis-specific assay and plan to use it to develop a multiplex immunofluorescence assay for simultaneous measurement of DNA damage and apoptotic markers on the same specimens.
There was significant within-treatment group variability for both biomarker levels (tumor and hair follicle) and tumor growth inhibition. We suspect that the within-group variability in response to exposure to topotecan and indenoisoquinolines in xenografts may, in part, be related to variable intratumoral levels of Top1 (38
). A number of tumor cell lines respond to Top1 inhibitors by selective S-phase arrest during DNA replication, and γH2AX formation in response to these treatments is restricted to replicating cells (19
). In addition, it has been reported in the FOCUS trial that response to irinotecan is a function of Top1 levels in the tumor at the start of treatment (40
). It is reasonable to suggest that the reduction in Top1 levels during and after Top1 inhibitor treatment can result in decreased drug response on the next treatment cycle. We have recently analytically validated an ELISA-based total Top1 assay in tissues (41
) and are interested in confirming the FOCUS trial results as well as documenting the effect of repeat Top1 inhibitor treatment on biomarker and xenograft growth response. The Top1 assay is being used as a correlative study in clinical trials at the NCI. We have also reported, using that assay, that there is significant variability in the amount of Top1 in xenografts from different mice, and that topotecan-sensitive xenografts express higher levels of Top1 protein than topotecan-resistant xenografts (38
The within-treatment group heterogeneity of γH2AX response measured in our preclinical model also suggests there might be similar variability in patient samples. To address this, the assay was designed to score γH2AX response based on the γH2AX background level above the patient’s pre-treatment biopsy sample background, with normalization to sample autofluorescence. Although it was not feasible to perform repeat sampling in the mouse model, the assay quantitation described here should allow each patient’s pre-dose sample to serve as a baseline readout and each sample to serve as its own control to offset inter- and intra-sample molecular and histologic heterogeneity. In addition, a calibrator panel and positive and negative control samples will be run during each clinical assay to ensure that quality control standards are met. It is expected that these quality control procedures will allow more accurate detection of a γH2AX response when applied to patient samples.
Use of hair follicles as a surrogate tissue for drug response was also examined, and data presented here suggest that γH2AX can be successfully measured in hair follicles following treatment in mouse models, albeit at higher doses of indenoisoquinolines and topotecan than required for response in xenografts. Hair follicle cells have long been recognized as a possible surrogate tissue for testing drug efficacy in patients undergoing chemotherapy; epithelial in origin, as are 80% of cancers, they can be obtained by noninvasive or minimally invasive procedures (44
). Hair follicle cells are among the most rapidly proliferating cells in the body and are therefore sensitive to agents that damage DNA. Other studies have demonstrated that antibody-based protein detection methods are effective in hair follicle cells (46
). One obvious limitation of our model is that decreased concentrations of the drugs across the surface area of the skin may result in higher doses being required to observe an effect in hair follicles. In addition, hair follicles are not abundant in athymic nude mice; additional studies are underway in rats and dogs to evaluate hair follicles as surrogate tissues. Because it was challenging to process individual hairs with intact follicles and sheaths on our automated tissue processing system, skin snips were collected to preserve the entire follicle. We identified the optimal clinical sampling time by determining that the mean number of nuclei per hair follicle positive for γH2AX in the A375 xenograft mice peaked 4 hours after treatment with topotecan and NSCs 724998 and 725776. Vehicle-treated animals generally had up to 5 positive nuclei per follicle (mean 3 positive nuclei per follicle), so the minimal cut-off for “positive” drug effect for clinical samples was set at 10 γH2AX-positive nuclei per follicle, or four standard deviations above the average vehicle response. A higher cut-off was selected due to the lack of published data regarding the amount of variability to be expected in specimens from patients with cancer, and may be adjusted after clinical trials with the indenoisoquinolines. Correlating γH2AX dose-response to Top1 inhibitors in hair follicles and tumor biopsies in clinical trials will establish the value of hair follicles as minimally invasive surrogates for drug effect in future trials of this class of drug.
Finally, a correlation between the γH2AX biomarker and effects on tumor growth was established at comparable dose levels in parallel experiments. However, the correlation of biomarker response to compound effect on tumor growth is fundamentally limited by the requirement for multiple administrations of any of the Top1 inhibitors we investigated to achieve xenograft growth suppression. In contrast, elevated γH2AX levels were observed within a few hours of a single administration of the compound. This rapid response to a single dose is a critical consideration in selecting a biomarker for use in the clinical population, where often only a single post-treatment biopsy is allowed.
In conclusion, γH2AX is a sensitive biomarker for monitoring the pharmacodynamics of anti-cancer therapeutics that damage DNA, and its measurement could lead to enhanced monitoring of chemotherapeutic effects in the clinic. Evaluation of γH2AX response in tumor biopsies or hair follicles allows for monitoring of pharmacodynamic effects in patients over the course of drug treatment. We anticipate that the use of this pharmacodynamic assay will inform clinical drug development decisions and promote the examination of surrogate tissues to evaluate the biochemical effects of Top1 inhibitors.
Statement of Translational Relevance
Optimal clinical development of a molecularly targeted agent requires the incorporation of validated pharmacodynamic assays to establish drug activity on target. This paper details the development and validation of a quantitative immunofluorescence assay for phosphorylated histone H2AX (γH2AX) as a biomarker for topoisomerase I (Top1) inhibitor activity. The assay was qualified in human tumor xenograft models using clinically relevant procedures and will be used in a phase I clinical trial conducted at the National Cancer Institute to evaluate the extent to which structurally similar indenoisoquinoline Top1 inhibitors activate γH2AX. In conjunction with toxicity and pharmacokinetic data, the pharmacodynamic results obtained will inform subsequent clinical development decisions. The γH2AX assay was also optimized to explore use of minimally invasive skin biopsies as surrogates for tumor biopsies; these studies will also be evaluated clinically and could have important implications for monitoring patients’ pharmacodynamic responses to chemotherapy with agents that damage DNA.