HHS Public Access Author manuscript Author Manuscript
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01. Published in final edited form as: Clin Cancer Res. 2016 July 1; 22(13): 3138–3147. doi:10.1158/1078-0432.CCR-16-0069.
Improving the Predictive Value of Preclinical Studies in Support of Radiotherapy Clinical Trials C. Norman Coleman1, Geoff S. Higgins2, J. Martin Brown3, Michael Baumann4, David G. Kirsch5, Henning Willers6, Pataje G.S. Prasanna1, Mark W. Dewhirst7, Eric J. Bernhard1, and Mansoor M. Ahmed1
Author Manuscript
1Radiation
Research Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, Maryland 2Cancer Research UK/ Medical Research Council, Oxford Institute for Radiation Oncology, University of Oxford, United Kingdom 3Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 4OncoRay National Center for Radiation Research, Technische Universität Dresden/ Helmholtz-Zenrtum Dresden-Rossendorf, Dresden, Germany and German Cancer Consortium, Dresden/ German Cancer Research Center (DKFZ) 5Departments of Radiation Oncology and Pharmacology and Cancer Biology, Duke University, Durham, North Carolina 6Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 7Departments of Radiation Oncology, Pathology and Biomedical Engineering, Duke University, Durham, North Carolina
Author Manuscript
Abstract
Author Manuscript
There is an urgent need to improve reproducibility and translatability of preclinical data in order to fully exploit opportunities for molecular therapeutics involving radiation and radio-chemotherapy. For in vitro the clonogenic assay remains the current state-of-the-art of preclinical assays, while newer moderate- and high-throughput assays offer the potential for rapid initial screening. Studies of radiation response modification by molecularly targeted agents can be improved using more physiologic 3D culture models. Elucidating effects on the cancer stem cells (CSC, and CSC-like) and developing biomarkers for defining targets and measuring responses are also important. In vivo studies are necessary to confirm in vitro findings, further define mechanism of action and address immune modulation and treatment-induced modification of the microenvironment. Newer in vivo models include genetically engineered and patient derived xenograft mouse models and spontaneously occurring cancers in domesticated animals. Selection of appropriate endpoints is important for in vivo studies, for example, regrowth delay measures bulk tumor killing while local tumor control assesses effects on CSC. The reliability of individual assays requires standardization of procedures and cross-laboratory validation. Radiation modifiers must be tested as part of clinical standard of care, which includes radio-chemotherapy for most tumors. Radiation models
Corresponding author: C. Norman Coleman, Radiation Research Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, 9609 Medical Center Drive, 3W102, Bethesda, MD 20892-9727. Phone: 240-276-5690; Fax: 240-276-5827; [email protected]. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Disclosure of potential conflicts of interest: No potential conflicts of interest were disclosed by the other authors.
Coleman et al.
Page 2
Author Manuscript
are compatible with, but also differ from those used for drug screening. Furthermore, the mechanism of a drug as a chemotherapy enhancer may be different than its interaction with radiation and/or radio-chemotherapy. This provides an opportunity to expand the use of moleculartargeted agents.
INTRODUCTION The era of personalized and precision medicine has emerged with a multitude of molecularly targeted drugs, immune modifiers and new classifications of cancer based on biologic/ genomic characteristics in addition to the organ of origin. Appropriate preclinical studies are critical to optimize targeted therapeutic strategies and clinical trial design to benefit the patients and also enhance the return on investment in translational research.
Author Manuscript
Approximately 60% of cancer patients in the developed world receive radiotherapy, often combined with systemic agents. Radiotherapy is a critical component of comprehensive cancer treatment in the developing world (1). It is effective and often curative, but would be more so if radiosensitizers, radioprotectors, and predictive biomarkers of patient and tumor radiation sensitivity were employed (2, 3). Radiation modifiers have been reviewed recently (4-8), as has the potential for radiation therapy to enhance the effectiveness of immunotherapeutics (9-11) to improve local tumor control as well as to induce abscopal effects that result in concomitant responses in distant metastases (12, 13).
Author Manuscript
One impediment to drug development has been irreproducibility of preclinical data (14). A 2012 NCI Radiation Research Program (RRP) workshop examined six randomized clinical trials from the Radiation Therapy Oncology Group that resulted in null outcomes (15). This result may be due in part to the quality and validity of the pre-clinical data. Stone et al. (16) after examining the details of 125 reported in vitro and in vivo preclinical studies on radiation-modifiers concluded that future preclinical studies must include: a) use of appropriate preclinical models that best represent the clinical setting of extant “standard-ofcare” multi-modality cancer therapy, b) fastidious calibration and dosimetry of radiation sources (17), c) detailed and accurate descriptions of experimental methodology and results, and d) clinically relevant drugs, doses, schedules, and assay conditions. Of critical importance is the recognition that the mechanism of action of a molecular-targeted drug may be different when used by itself to target a specific tumor pathway compared to how it affects combination therapy that includes radiation.
Author Manuscript
Here we review issues that could enhance the strength of preclinical models of radiationmodifying drugs in leading to early phase clinical trials. The perspective includes specific aspects of in vitro assay systems and in vivo rodent and companion canine tumor models with the goal of identifying critical steps, gaps and new approaches toward enhancing the use of rapidly emerging discoveries in cancer treatment with radiation therapy. Recommendations are presented with the recognition of substantial ongoing changes in cell lines and models for screening drugs for cancer treatment moving from the NCI- 60 cell lines (18) to patient derived xenografts (PDX) (19). While the radiation-modifying drugs could enhance the immune response through the radiation-induced changes in the tumor cell, this review does not include discussion of models for screening and assessing Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 3
Author Manuscript
immunotherapy. Radiation-induced immune-modulation is a major topic in itself now under active development including novel animal models with functioning humanized immune systems (20).
FOCUS AREAS: PRECLINICAL RADIOBIOLOGICAL ASSAYS
Author Manuscript
Key components of the workflow of pre-clinical radiobiological assays (Figure 1A) and potential avenues to enhance standards and quality (Figure 1B) are discussed. There is general agreement that the ability of a treated cell to form a colony, as measured by the “clonogenic assay” (21), should be considered the “gold standard” for testing radiation modifiers, although a number of commercial and short-term assays likely will be required for initial screening of drugs followed by clonogenic assays for confirmation of results wherever possible. The clonogenic assay does not work for all cell lines and this is currently problematic for patient derived in vitro models (18, 22, 23). In the future it is envisioned that short term assays using patient-derived cell lines and 3D models will be benchmarked against the gold standard clonogenic assay, using cell lines that are amenable to this endpoint. In-Vitro Radiosensitivity Screening Assays Higgins et al. (24) reported two types of screening assays for determining new radiosensitization targets including a DNA damage readout and a medium/high throughput clonogenic assay. The DNA damage readout assay (Figure 2A) was used with a siRNA library targeted against 200 DNA Damage Response (DDR) genes. Screening of both tumor and normal cell lines identified polymerase theta (POLQ) as a tumor specific radiosensitization target.
Author Manuscript
The medium/high throughput clonogenic assay (Figure 2B) uses an automated 96-well plate colony counting platform with the cell kill measurable to 2 logs (0.01) surviving fraction (25). Screening 10,000 genes in HeLa cells in 8 months identified several new targets. One of these is Thiamin pyrophosphokinase 1 (TPK1). The radiosensitizing effects of TPK1 knockdown was subsequently investigated in several other cell lines and confirmed to radiosensitize tumor but not normal cell lines. Work in progress includes evaluating suitability of other cells for this assay system, using drugs in addition to gene knockdowns and validation of hits in in vivo models.
Author Manuscript
Lin et al. (26) describe a novel High Content Clonogenic Survival Assay that is similar in concept to that of Higgins et al. (24). This assay performed well compared to traditional clonogenic assay with an upper radiation dose limit of 6 Gy (26). This assay was used in a drug screen to identify trametinib as a sensitizer for K-RAS mutant tumor cells. Like the assay of Higgins et al. (24), the range of SF is limited to 2 logs of kill, but notably, both measure cell killing rather than inhibition of proliferation as in most in short-term viability/ proliferation assays. Liu et al. reported an automated high-throughput (HTS) drug-screening platform (Figure 3) (27) based on short-term cell proliferation/survival assays (3-5 days incubation). These assays were used to derive a Short-term Radiosensitization Factor at 2 Gy (SRF2Gy), which
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 4
Author Manuscript
correlates with standard Dose Enhancement Factors (DEF) derived from clonogenic survival assays using radiation doses up to 8 Gy. The correlation between the SRF2Gy and the DEF at SF of 0.1 (DEFSF0.1) had a sensitivity and specificity of >80%. Drug-induced short-term radiosensitization was accompanied by changes in the mode of cell death such as senescence or apoptosis, thus providing a mechanistic basis as to why short-term endpoints could effectively predict radiosensitization in a clonogenic assay. The results obtained with this assay underscored the impact of genomic heterogeneity among tumors and the need to examine the activity of a targeted drug in a broad range of tumors. Further development of this automated platform would allow high-throughput initial screening of drugs against tumors from a range of established and patient-derived cell lines with defined genomic backgrounds for establishing drug-biomarker-radiosensitization relationships. In-Vitro: Endpoints and 3D Models
Author Manuscript
Short-term assay endpoints include growth inhibition (analysis of cell number after 2-4 days of exposure to an agent by assays such as MTT or XTT), cell viability analysis (trypan blue exclusion), and measures of apoptosis. The results of short-term assays do not always correlate with long-term assays that measure tumor clonogens (clonogenic assay and human tumor stem cell assays). For example, Brown and Wouters showed that while differences in genetic makeup (p53 mutation status) can change the frequency of apoptosis (a short-term assay) induced by radiation or chemotherapy (28), it plays little or no role in clonogenic survival that captures all mechanisms of cell death (29). In contrast, a positive impact of p53 mutation on increased cell sensitivity to DNA damaging agents was detected by short-term assay in the NCI-60 cell line panel (30). Thus it is important to understand the mechanism by which cancer cells die (31), and its impact on the endpoint measured in the various assays.
Author Manuscript Author Manuscript
There are significant differences between 2D and 3D cell culture systems. For example, Eke et al. showed that cells cultured in a 3D extracellular matrix are more radio- and chemoresistant compared to cells grown under 2D cell culture conditions (32). A co-culture assay platform was developed for drug screening via high-content analysis that could further mimic in vivo systems (33). Our conclusion is that clonogenic or similar long-term assays are essential tools for validating and standardizing the testing of radiation modifiers in HTS assays. While HTS is feasible with certain cells using a clonogenic survival endpoint, patient-derived tumor models will likely require non-clonogenic endpoints. 3D systems including those with extracellular matrix and with co-culture with normal cells represent approaches for initial screening of a limited number of compounds as well as for secondary screens of lead compounds identified in initial 2D screens. Validating the effects of novel radiation modifiers by examining their effects on a relevant panel of tumor and normal cell lines is key to establishing their clinical relevance. No in vitro cell culture assays will be able to fully assess the efficacy of agents that work through the microenvironment such as improved tumor oxygenation, altered immune cell infiltration or altered tumor vasculature.
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 5
In-Vivo: Approaches
Author Manuscript Author Manuscript Author Manuscript
Tumor Control Dose (TCD50) assay—Baumann and Krause (34) have reported extensively on the use of Tumor Control Dose (TCD50) assay (i.e., the radiation dose that results in a 50% probability of tumor control) as a cancer stem cell (CSC, also referred to as tumor-initiating cells or CSC-like) related endpoint in preclinical tumor models. Data from Hill and Milas (35) demonstrated the importance of CSC number and tumor control in the TCD50 assay (34) (Figure 4). They demonstrated an inverse linear correlation between their TD50 (Take Dose 50, i.e., the cell number necessary to achieve a tumor growth in 50% of the host animals) and the TCD50 after single-dose irradiation under homogeneous hypoxia (35-37). The importance of inactivation of CSC by radiotherapy for local tumor control is supported by a number of further experiments (38) and recent clinical data, for example, for patients with early stage laryngeal cancer using the putative CSC marker CD44 (36, 39), CSC number correlated with permanent local tumor control. In line with this result, a multicenter retrospective analysis of postoperative radiochemotherapy in HPV-negative high-risk HNSCC revealed a significant correlation between high levels of CD44 protein expression as well as mRNA expressions of other putative CSC markers and low locoregional tumor control after radiochemotherapy (40). A further example is the observation that low 26S proteasome activity, which leads to CSC-like phenotype with higher self-renewal capacity and higher tumorigenicity, correlates with locoregional control in HNSCC (41). Similar correlations have also been found for other tumor types investigated using a variety of putative CSC markers (42). It is important to note that so-called CSC markers do not directly indicate a single CSC, but rather correlate with enrichment of CSCs in a subpopulation. In addition, remarkable plasticity has been recognized between non-CSC and CSC phenotype in tumor cells (43, 44). For example, therapeutic interventions including fractionated irradiation may lead to epigenetic reprogramming of non-CSC to CSC-like tumor cells (45). Despite these important limitations, the clinical and preclinical observations described above indicate that surrogate markers of CSC density and number (36) are significantly correlated with the probability of local tumor control after radiotherapy, supporting the need for in vivo assays to reflect CSC inactivation.
Author Manuscript
Tumor growth delay assays may not always concur with the TCD50 assays (46). However, growth delay experiments are useful for gaining mechanistic insights and also because shrinkage and growth delay provide clinical benefit. Thus, an unresolved issue is whether there is an optimal series of in vivo assay(s) that should be done prior to proposing a clinical trial. TCD50 assays are performed much less commonly than growth delay assays, because of the larger number of animals and higher cost. Nevertheless, before clinical trials with curative endpoints are initiated, TCD50 assays, which better reflect CSC inactivation, should be considered to reduce the chance of a negative trial. This may require co-operation between specialized laboratories for practical reasons. While the TCD50 assay has been widely used in radiotherapy studies, the same arguments for their use can also be made for other therapeutic interventions when their curative potential is tested (38) Genetically engineered mouse models (GEMM)—GEMMs allow for the generation of primary tumors that develop in a mouse with an intact immune system and within a native microenvironment. Therefore, GEMMs may be particularly useful in
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 6
Author Manuscript
modeling the response of cancer to radiation and immunotherapy. Because GEMM cancers can be generated in a temporally and spatially restricted manner (47, 48), these models can be used to study the response of cancer to chemo-radiotherapy in vivo. One of the most attractive features of GEMMs is the ability to use different recombinases to mutate genes in specific cell types for mechanistic studies. Kirsch et al. (48) have used a dual recombinase GEMM to answer a fundamental question in radiobiology: Is tumor sensitivity defined by the tumor cell or host stromal components? Figure 5 outlines the results of these studies published in Moding et al. (49). With the development of Flp-regulated CreER alleles (50), dual recombinase technology can also be applied for sequential mutagenesis within tumor cells to investigate mechanisms of tumor maintenance and response to radiation therapy. These approaches could be expanded to include newer gene technologies such as CRISPR/ Cas9.
Author Manuscript
Other animal models—Companion animal studies, primarily in dogs, are a focus of the Comparative Oncology Program at the NCI. Increased interest in these models is highlighted in a recent report by the Institute of Medicine, summarizing a 2-day workshop held in 2015. A summary of this conference has recently been reported (51). These offer unique opportunities for drug and chemo-radiotherapy development (52). Canine tumors are genetically diverse, grow relatively slowly, exhibit spatio-temporal heterogeneity and diversity, are amenable to imaging studies, and can help assess cost-effective treatment choices (53). Additionally, they are not subject to HIPAA regulations, allowing for greater flexibility in conducting retrospective tissue analyses. An example of the type of data from a canine trial is provided in Supplementary Figure S1. In-Vivo: Biomarkers and Other Endpoints
Author Manuscript Author Manuscript
Other potential confounders and considerations should be taken into account when using the results of a screening assay for a combined radio-chemotherapy clinical trial. These include: a) the differential impact of the drug on the CSC and non-CSC compartments, b) the effect of the tumor microenvironment on radiation response (e.g., hypoxia) or the other components of the microenvironment as noted above, and c) the fate of various cell populations following fractionated irradiation. The impact of multi-fraction irradiation on altering the cellular molecular phenotype has been reviewed by Makinde et al. (54). Radiation-inducible targets may be exploitable for immunotherapy and molecular-targeted therapy. A comprehensive discussion of biomarkers is beyond the scope of this article, but biomarker development is an essential part of drug development and treatment selection. The γ-H2AX biomarker is frequently used for assessing the impact of therapy (55). Other DNA damage and tumor biomarkers will provide enhanced and potentially early predictive information. Opportunities exist to use tumor radiation resistance/sensitivity biomarkers to escalate/de-escalate radiation dose and to spare normal tissue toxicity. Biomarkers that assess tumor characteristics are being used for making treatment choices in clinical trials (56-58).
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 7
Author Manuscript
OTHER CONSIDERATIONS In addition to the radiobiological issues outlined above, there are additional general points to consider. Gene expression patterns can vary based on the location of the tumor cells in a tissue culture or the location of implantation in a rodent model - xenograft vs. orthotopic (59). Gene silencing is often used to study the effect of targeting a specific molecular pathway with drug(s) in an experimental model. However, John-Aryankalayil et al. showed that the gene expression pattern can be substantially different when the target gene is silenced in comparison to that seen when a pathway is drugged and also that drug concentrations in excess of what is clinically relevant can lead to mechanisms of action that may be irrelevant in the clinic (60).
Author Manuscript Author Manuscript
Spatio-temporal heterogeneity within tumors during tumor evolution, at the time of initial biopsy and profiling, and over time during and after treatment is another key and highly complex consideration. The provocative report in renal cell cancer by Gerlinger et al. demonstrated the heterogeneity in tumors present de novo and the likelihood of adaptation/ evolution during therapy (25). The complexity of heterogeneity includes genomic heterogeneity, epigenetic changes in tumor phenotype, environmental and metabolic influences on treatment outcome, normal tissue-tumor cell interaction, including cell-to-cell signaling, and immunological responses (61). In vivo organoid models (62) may mimic the hypoxic aspects of heterogeneity as the 3D models (32, 33) can reproduce cell-matrix interaction. The move to PDX addresses certain aspects of heterogeneity (63) but many important factors relating to treatment outcome as well as evolution under the treatment conditions will not be accounted for in a pre-treatment xenograft (64, 65). While speculative, given the ability to target radiation dose to specific volumes, radiotherapy may be a very useful “drug” with which to alter tumor behavior in a way that makes treatments more effective, in addition to the cell killing by radiation itself (54, 66).
TOWARD IMPROVED PRE-CLINICAL MODELS OF RADIATION MODIFIERS
Author Manuscript
A pathway for the development of radiation modifiers published recently (8, 67) suggests modifications to address the new biology; however time-honored assessments often referred to pejoratively as “classical” remain critical to both drug and radiation clinical trials (66). While animal normal tissue models may provide only limited information, normal tissue injury remains an integral part of preclinical development (67, 68) The need for studies will depend on the size of the treatment field as highly focused radiation fields in some regimens are designed to be at tissue-damaging doses to limited volumes and thereby minimizing clinically significant late effects. Critical issues raised about translation of pre-clinical study results to clinical trials are being addressed (69, 70). Recognizing the rapid evolution in tumor biology and model systems and our reluctance to be prescriptive in such a complex field, a general approach to preclinical assessment of radiation modifiers may be as follows (see Figure 1A).
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 8
Author Manuscript
Step 1. “HTS” screens need to be harmonized for 2D cultures so that comparable results can reliably obtained by different laboratories. The techniques themselves can be varied as long as the results are reasonably compatible and/or differences understood. In settings in which a limited number of compounds are being tested, the initial screening may be Step 2.
Author Manuscript
Step 2. When clonogenic assays are possible, these should be conducted as they measure cell killing down to 3-4 log kill and not just growth inhibition. Notably, few drugs are subject to a clonogenic assay and/or cell survival below 1 log is rarely assessed in short-term viability/ proliferation assay. In vitro assays should consider CSC-like cells that are relevant for radioresistance, for example through the use of tumor spheres. Further, 3D models can help approximate some microenvironmental factors. Prior to going into animal models, Step 2 may also include assessing the impact of concurrently given chemotherapeutics on radiosensitizing drug effects. The inclusion of patient-derived tumor models is recommended if the initial screen was done on established cancer cell lines. In general, Step 2 narrows down the number compounds that will undergo the next step in testing. Step 3. Assessment of efficacy in murine models should include treatment regimens that are clinically relevant. This will include combinations of radiation plus drugs that are the standard of care and an appropriate radiation schedule. While regrowth delay assays may not necessarily correspond to local tumor control, they are useful for evaluating drug-radiation interaction, mechanisms of action and microenvironmental effects. The initial models may be xenografts as are now employed or newer models from patient-derived tumors with the models selected based on their utility for predicting clinical results (16).
Author Manuscript
Step 4. At this point, expansion of in vivo systems beyond growth delay should be selectively applied based on the specific mechanistic issues being addressed and also on the aim of assessing local control as a clinically relevant endpoint, such as with the TCD50 assay, GEMMs can help determine mechanisms of action and which cells are the “target”, and companion canine models have all the components of spontaneous human tumors, as well as providing needed care to the animals. It may also be equally important to study normal tissue toxicity to critical organs in tumor bearing animal models, to determine the overall risk-benefit ratio.
Author Manuscript
Figure 1 summarizes the work flow sequence for radiation modifier preclinical development and also the hierarchy of steps to be followed with increasingly stringent laboratory protocols for preclinical radiobiology assays to ensure rigorous quality control, assure validity of pre-clinical data to conduct clinical studies and then provide broad application beyond the single laboratory that developed the assay. Such attention to detail is a key issue for the Food and Drug Administration for Laboratory Developed tests (71).
CONCLUSIONS Rapid advances in cancer biology are uncovering many potential targets for cancer treatment. This has led to a need to screen potential drugs/treatments with accuracy and efficiency so that subsequent assessments have higher chances of choosing effective
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 9
Author Manuscript
treatments. Establishing biomarkers that can be used to select the optimal treatment and monitor its effects will facilitate this process. Cancer researchers and physicians, while working to improve the knowledge in their specific fields and enhance the efficacy and reduce normal tissue toxicity need to be cognizant that the best treatment for a given patient may not necessarily be contained within their particular area of interest/research focus. To help them in treatment selection, results across preclinical studies need to be comparable, which requires fastidious assay development and crossplatform comparisons. In this context we stress the continued importance of well-done preclinical trials that are comparable between laboratories and platforms. The continued utility of “classic” methodologies in chemotherapy and radiation therapy including using clinically relevant drug doses, schedules, and assay conditions should not be overlooked in these trials.
Author Manuscript
It is expected that the relationship between the “new biology” and advancing clinical care will involve complex multi-modality care requiring knowledge of the initial tumor characteristics, biomarkers of tumor response and adaptation to treatment before treatment resistance arises. The potential for the use of radiation therapy as curative and palliative treatment (1) provides unique opportunities to exploit the technological advances in radiation together with molecular-targeted and immune-modulating agents. There is potential to use a spectrum of radiation treatments including highly focused, broad field/ whole body or systemic radiation at specified doses and dose rates, which will open up possibilities for combination treatments requiring careful development, implementation, and ongoing assessment. These are the “next steps” for novel biologically-based radiation oncology.
Author Manuscript
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments M. Baumann reports receiving a commercial research grant from Merck. D.G. Kirsch reports receiving a commercial research grant from GlaxoSmithKline. M.W. Dewhirst reports receiving commercial research grants from Aerpio Therapeutics, BioMimetix Pharmaceutical, and Janssen; has ownership interest in Celsion; and was a consultant/advisory board member for EMD Serono. Grant Support: H. Willers is supported by the American Cancer Society (123420RSG-12-224-01-DMC).
Author Manuscript
REFERENCES 1. Atun R, Jaffray DA, Barton MB, Bray F, Baumann M, Vikram B, et al. Expanding global access to radiotherapy. Lancet Oncol. 2015; 16:1153–86. [PubMed: 26419354] 2. Begg AC, Stewart FA, Vens C. Strategies to improve radiotherapy with targeted drugs. Nat Rev Cancer. 2011; 11:239–53. [PubMed: 21430696] 3. Schaue D, McBride WH. Opportunities and challenges of radiotherapy for treating cancer. Nat Rev Clin Oncol. 2015; 12:527–40. [PubMed: 26122185]
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 10
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
4. Higgins GS, O'Cathail SM, Muschel RJ, McKenna WG. Drug radiotherapy combinations: review of previous failures and reasons for future optimism. Cancer Treat Rev. 2015; 41:105–13. [PubMed: 25579753] 5. Liauw SL, Connell PP, Weichselbaum RR. New paradigms and future challenges in radiation oncology: an update of biological targets and technology. Sci Transl Med. 2013; 5:173sr2. [PubMed: 23427246] 6. Morgan MA, Parsels LA, Maybaum J, Lawrence TS. Improving the efficacy of chemoradiation with targeted agents. Cancer Discov. 2014; 4:280–91. [PubMed: 24550033] 7. Le QT, Shirato H, Giaccia AJ, Koong AC. Emerging treatment paradigms in radiation oncology. Clin Cancer Res. 2015; 21:3393–401. [PubMed: 25991820] 8. Prasanna PG, Narayanan D, Hallett K, Bernhard EJ, Ahmed MM, Evans G, et al. Radioprotectors and radiomitigators for improving radiation therapy: The Small Business Innovation Research (SBIR) gateway for accelerating clinical translation. Radiat Res. 2015; 184:235–48. [PubMed: 26284423] 9. Demaria S, Golden EB, Formenti SC. Role of local radiation therapy in cancer immunotherapy. JAMA Oncol. 2015; 1:1325–32. [PubMed: 26270858] 10. Ahmed MM, Hodge JW, Guha C, Bernhard EJ, Vikram B, Coleman CN. Harnessing the potential of radiation-induced immune modulation for cancer therapy. Cancer Immunol Res. 2013; 1:280–4. [PubMed: 24777964] 11. Ahmed MM, Guha C, Hodge JW, Jaffee E. Immunobiology of radiotherapy: new paradigms. Radiat Res. 2014; 182:123–5. [PubMed: 25036983] 12. Chandra RA, Wilhite TJ, Balboni TA, Alexander BM, Spektor A, Ott PA, et al. A systematic evaluation of abscopal responses following radiotherapy in patients with metastatic melanoma treated with ipilimumab. Oncoimmunology. 2015; 4:e1046028. [PubMed: 26451318] 13. Vanpouille-Box C, Pilones KA, Wennerberg E, Formenti SC, Demaria S. In situ vaccination by radiotherapy to improve responses to anti-CTLA-4 treatment. Vaccine. 2015; 33:7415–22. [PubMed: 26148880] 14. Challenges in Irreproducible Research [about 3 screens]. [cited 2015 Dec 15]. Available from: http://www.nature.com/news/reproducibility-1.17552 15. Liu FF, Okunieff P, Bernhard EJ, Stone HB, Yoo S, Coleman CN, et al. Lessons learned from radiation oncology clinical trials. Clin Cancer Res. 2013; 19:6089–100. [PubMed: 24043463] 16. Stone HB, Coleman CN, Deye J, Capala J, Mitchell JB, Martin JB. Preclinical data on efficacy of 10 drug-radiation combinations: evaluations, concerns and recommendations. Transl Oncol. 2016; 9:46–56. [PubMed: 26947881] 17. Desrosiers M, DeWerd L, Deye J, Lindsay P, Murphy MK, Mitch M, et al. The importance of dosimetry standardization in radiobiology. J Res Natl Inst Stand Technol. 2013; 118:403–18. [PubMed: 26401441] 18. Chabner BA. NCI-60 cell line screening: a radical departure in its time. J Natl Cancer Inst. 2016; 108:djv388. [PubMed: 26755050] 19. Ledford H. US cancer institute to overhaul tumour cell lines. Nature. 2016; 530:391. [PubMed: 26911756] 20. Sanmamed MF, Chester C, Melero I, Kohrt H. Defining the optimal murine models to investigate immune checkpoint blockers and their combination with other immunotherapies. Ann Oncol. Feb 23.2016 [Epub ahead of print]. 21. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006; 1:2315–9. [PubMed: 17406473] 22. van de Wetering M, Francies HE, Francis JM, Bounova G, Iorio F, Pronk A, et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell. 2015; 161:933–45. [PubMed: 25957691] 23. Crystal AS, Shaw AT, Sequist LV, Friboulet L, Niederst MJ, Lockerman EL, et al. Patient-derived models of acquired resistance can identify effective drug combinations for cancer. Science. 2014; 346:1480–6. [PubMed: 25394791]
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 11
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
24. Higgins GS, Prevo R, Lee YF, Helleday T, Muschel RJ, Taylor S, et al. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown. Cancer Res. 2010; 70:2984–93. [PubMed: 20233878] 25. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012; 366:883–92. [PubMed: 22397650] 26. Lin SH, Zhang J, Giri U, Stephan C, Sobieski M, Zhong L, et al. A high content clonogenic survival drug screen identifies MEK inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer. J Thorac Oncol. 2014; 9:965–73. [PubMed: 24922006] 27. Liu Q, Wang M, Kern AM, Khaled S, Han J, Yeap BY, et al. Adapting a drug screening platform to discover associations of molecular targeted radiosensitizers with genomic biomarkers. Mol Cancer Res. 2015; 13:713–20. [PubMed: 25667133] 28. Brown JM, Wouters BG. Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res. 1999; 59:1391–9. [PubMed: 10197600] 29. Brown JM, Wouters BG, Kirsch DG. Cell death identification in anticancer therapy- Letter. Cancer Res. 2015; 75:3681. [PubMed: 26286478] 30. O'Connor PM, Jackman J, Bae I, Myers TG, Fan S, Mutoh M, et al. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res. 1997; 57:4285–300. [PubMed: 9331090] 31. Rello-Varona S, Herrero-Martin D, Lopez-Alemany R, Munoz-Pinedo C, Tirado OM. "(Not) all (dead) things share the same breath": identification of cell death mechanisms in anticancer therapy. Cancer Res. 2015; 75:913–7. [PubMed: 25724677] 32. Eke I, Cordes N. Radiobiology goes 3D: how ECM and cell morphology impact on cell survival after irradiation. Radiother Oncol. 2011; 99:271–8. [PubMed: 21704412] 33. Krausz E, de Hoogt R, Gustin E, Cornelissen F, Grand-Perret T, Janssen L, et al. Translation of a tumor microenvironment mimicking 3D tumor growth co-culture assay platform to high-content screening. J Biomol Screen. 2013; 18:54–66. [PubMed: 22923784] 34. Baumann, M.; Krause, M. Tumor biology's impact on clinical cure rates. In: Molls, M.; Vaupel, P.; Nieder, C.; Anscher, MS., editors. The impact of tumor biology on cancer treatment and multidisciplinary strategies. Springer-Verlag; Berlin, Heidelberg (Germany): 2009. p. 323-34. 35. Hill RP, Milas L. The proportion of stem cells in murine tumors. Int J Radiat Oncol Biol Phys. 1989; 16:513–8. [PubMed: 2921157] 36. Baumann M, Krause M. CD44: a cancer stem cell-related biomarker with predictive potential for radiotherapy. Clin Cancer Res. 2010; 16:5091–3. [PubMed: 20861165] 37. Krause M, Yaromina A, Eicheler W, Koch U, Baumann M. Cancer stem cells: targets and potential biomarkers for radiotherapy. Clin Cancer Res. 2011; 17:7224–9. [PubMed: 21976536] 38. Baumann M, Krause M, Hill R. Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer. 2008; 8:545–54. [PubMed: 18511937] 39. de Jong MC, Pramana J, van der Wal JE, Lacko M, Peutz-Kootstra CJ, de Jong JM, et al. CD44 expression predicts local recurrence after radiotherapy in larynx cancer. Clin Cancer Res. 2010; 16:5329–38. [PubMed: 20837694] 40. Linge A, Lock S, Gudziol V, Nowak A, Lohaus F, von Neubeck C, et al. Low cancer stem cell marker expression and low hypoxia identify good prognosis subgroups in HPV(−) HNSCC after postoperative radiochemotherapy: a multicenter study of the DKTK-ROG. Clin Cancer Res. Jan 11.2016 [Epub ahead of print]. 41. Lagadec C, Vlashi E, Bhuta S, Lai C, Mischel P, Werner M, et al. Tumor cells with low proteasome subunit expression predict overall survival in head and neck cancer patients. BMC Cance. 2014; 14:152. 42. Butof R, Dubrovska A, Baumann M. Clinical perspectives of cancer stem cell research in radiation oncology. Radiother Oncol. 2013; 108:388–96. [PubMed: 23830466] 43. Kreso A, Dick JE. Evolution of the cancer stem cell model. Cell Stem Cell. 2014; 14:275–91. [PubMed: 24607403]
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 12
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
44. Vlashi E, Pajonk F. Cancer stem cells, cancer cell plasticity and radiation therapy. Semin Cancer Biol. 2015; 31:28–35. [PubMed: 25025713] 45. Cojoc M, Peitzsch C, Kurth I, Trautmann F, Kunz-Schughart LA, Telegeev GD, et al. Aldehyde dehydrogenase is regulated by beta-Catenin/TCF and promotes radioresistance in prostate cancer progenitor cells. Cancer Res. 2015; 75:1482–94. [PubMed: 25670168] 46. Budach W, Budach V, Stuschke M, Dinges S, Sack H. The TCD50 and regrowth delay assay in human tumor xenografts: differences and implications. Int J Radiat Oncol Biol Phys. 1993; 25:259–68. [PubMed: 8420873] 47. Lee CL, Moding EJ, Huang X, Li Y, Woodlief LZ, Rodrigues RC, et al. Generation of primary tumors with Flp recombinase in FRT-flanked p53 mice. Dis Model Mech. 2012; 5:397–402. [PubMed: 22228755] 48. Kirsch DG, Dinulescu DM, Miller JB, Grimm J, Santiago PM, Young NP, et al. A spatially and temporally restricted mouse model of soft tissue sarcoma. Nat Med. 2007; 13:992–7. [PubMed: 17676052] 49. Moding EJ, Lee CL, Castle KD, Oh P, Mao L, Zha S, et al. ATM deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium. J Clin Invest. 2014; 124:3325–38. [PubMed: 25036710] 50. Zhang M, Kirsch DG. The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis. Dis Model Mech. 2015; 8:1155–66. [PubMed: 26183214] 51. LeBlanc AK, Breen M, Choyke P, Dewhirst M, Fan TM, Gustafson DL, et al. Perspectives from man's best friend: National Academy of Medicine's Workshop on Comparative Oncology. Sci Transl Med. 2016; 8:324. 52. LeBlanc AK, Mazcko CN, Khanna C. Defining the value of a comparative approach to cancer drug development. Clin Cancer Res. Dec 28.2015 [Epub ahead of print]. 53. Chi JT, Thrall DE, Jiang C, Snyder S, Fels D, Landon C, et al. Comparison of genomics and functional imaging from canine sarcomas treated with thermoradiotherapy predicts therapeutic response and identifies combination therapeutics. Clin Cancer Res. 2011; 17:2549–60. [PubMed: 21292819] 54. Makinde AY, John-Aryankalayil M, Palayoor ST, Cerna D, Coleman CN. Radiation survivors: understanding and exploiting the phenotype following fractionated radiation therapy. Mol Cancer Res. 2013; 11:5–12. [PubMed: 23175523] 55. Koch U, Hohne K, von Neubeck C, Thames HD, Yaromina A, Dahm-Daphi J, et al. Residual gamma H2AX foci predict local tumour control after radiotherapy. Radiother Oncol. 2013; 108:434–9. [PubMed: 23891089] 56. Redig AJ, Janne PA. Basket trials and the evolution of clinical trial design in an era of genomic medicine. J Clin Oncol. 2015; 33:975–7. [PubMed: 25667288] 57. NCI-Molecular Analysis for Therapy Choice (NCI-MATCH) Trial [about 9 screens]. [cited 2015 Dec 31]. Available from: http://www.cancer.gov/about-cancer/treatment/clinical-trials/ncisupported/nci-match 58. Baumann M, Krause M, Overgaard J, Debus J, Bentzen SM, Daartz J, et al. Radiation oncology in the era of precision medicine. Nat Rev Cancer. 2016; 16:234–49. [PubMed: 27009394] 59. Kahn J, Tofilon PJ, Camphausen K. Preclinical models in radiation oncology. Radiat Oncol. 2012; 7:223. [PubMed: 23270380] 60. John-Aryankalayil M, Palayoor ST, Cerna D, Falduto MT, Magnuson SR, Coleman CN. NS-398, ibuprofen, and cyclooxygenase-2 RNA interference produce significantly different gene expression profiles in prostate cancer cells. Mol Cancer Ther. 2009; 8:261–73. [PubMed: 19139136] 61. Alizadeh AA, Aranda V, Bardelli A, Blanpain C, Bock C, Borowski C, et al. Toward understanding and exploiting tumor heterogeneity. Nat Med. 2015; 21:846–53. [PubMed: 26248267] 62. Hubert CG, Rivera M, Spangler LC, Wu Q, Mack SC, Prager BC, et al. A three-dimensional organoid culture system derived from human glioblastomas recapitulates the hypoxic gradients and cancer stem cell heterogeneity of tumors found in vivo. Cancer Res. Feb 19.2016 [Epub ahead of print].
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 13
Author Manuscript Author Manuscript
63. Cassidy JW, Caldas C, Bruna A. Maintaining tumor heterogeneity in patient-derived tumor xenografts. Cancer Res. 2015; 75:2963–8. [PubMed: 26180079] 64. Siolas D, Hannon GJ. Patient-derived tumor xenografts: transforming clinical samples into mouse models. Cancer Res. 2013; 73:5315–9. [PubMed: 23733750] 65. Gao H, Korn JM, Ferretti S, Monahan JE, Wang Y, Singh M, et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nat Med. 2015; 21:1318–25. [PubMed: 26479923] 66. Coleman CN, Lawrence TS, Kirsch DG. Enhancing the efficacy of radiation therapy: premises, promises, and practicality. J Clin Oncol. 2014; 32:2832–5. [PubMed: 25113766] 67. Lawrence YR, Vikram B, Dignam JJ, Chakravarti A, Machtay M, Freidlin B, et al. NCI-RTOG translational program strategic guidelines for the early-stage development of radiosensitizers. J Natl Cancer Inst. 2013; 105:11–24. [PubMed: 23231975] 68. Harrington KJ, Billingham LJ, Brunner TB, Burnet NG, Chan CS, Hoskin P, et al. Guidelines for preclinical and early phase clinical assessment of novel radiosensitisers. Br J Cancer. 2011; 105:628–39. [PubMed: 21772330] 69. Landis SC, Amara SG, Asadullah K, Austin CP, Blumenstein R, Bradley EW, et al. A call for transparent reporting to optimize the predictive value of preclinical research. Nature. 2012; 490:187–91. [PubMed: 23060188] 70. Funding Opportunity Announcement: Cooperative Agreement to Develop Targeted Agents for Use with Systemic Agents Plus Radiotherapy (U01) [about 19 screens]. [cited 2016 Mar 7]. Available from: http://grants.nih.gov/grants/guide/pa-files/PAR-16-111.html 71. Laboratory Developed Tests [about 4 screens]. [cited 2015 Dec 15]. Available from: http:// www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ ucm407296.htm 72. Moding EJ, Castle KD, Perez BA, Oh P, Min HD, Norris H, et al. Tumor cells, but not endothelial cells, mediate eradication of primary sarcomas by stereotactic body radiation therapy. Sci Transl Med. 2015; 7:278.
Author Manuscript Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 14
Author Manuscript Author Manuscript
Figure 0001
Author Manuscript Author Manuscript
Figure 0002 Figure 1. A. Workflow of pre-clinical radiobiology experimental tasks from the discovery phase to clinical trials
The critical steps are the clonogenic assay and regrowth delay assay using clinically relevant model that includes the extant standard of care that may include systemic agents. Initial drug screening may be high/moderate throughput automated assays or more targeted clonogenic screening. The options for in vivo assays include mouse xenograft models and patient derived assays. Additional studies may be needed to address mechanism of action, normal Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 15
Author Manuscript
tissue toxicity, target cells and microenvironment, such as TCD50 using GEMMs, or canine companion models. B. Steps for assay development to improve quality control and quality assurance for the preclinical radiobiology assays. Developing and standardizing assay procedures are critical for the broad clinical applicability of the assays beyond the research laboratories in which the concept is initially discovered and developed. GEMM, genetically engineered mouse models; PDX, patient derived xenograft; TCD50, Tumor control dose50.
Author Manuscript Author Manuscript Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 16
Author Manuscript Author Manuscript
Figure 0003
Author Manuscript Figure 0004
Author Manuscript
Figure 2. Moderate-/high throughput screening radiosensitivity assay and protocol
A. High throughput screening (HTS) for new radiation sensitizer targets. Cells are transfected in quadruplicate with half the plates receiving 4-Gy radiation. At 24 h plates are stained for γH2AX and read with a reader. B. Medium throughput clonogenic assay. NT, nontargeting.
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 17
Author Manuscript Author Manuscript
Figure 3. The automated drug discovery platform at the Massachusetts General Hospital Cancer Center (MGHCC)
Illustration of an automated robotic drug-screening platform to discover associations of molecular targeted radiosensitizers with genomic biomarkers. Adapted from Liu and colleagues (27). GI, gastrointestinal; GU, genitourinary; HNSCC, head and neck small cell cancer; SRF, short-term radiosensitization factor.
Author Manuscript Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 18
Author Manuscript Author Manuscript
Figure 4. Tumor control dose (TCD50) assay
Author Manuscript
A. Local tumor control depends on cancer stem cell inactivation and should therefore have an important role in preclinical in vivo assays. B. The response to radiotherapy of tumors of the same entity and size differs due to intertumoral heterogeneity in (radio)biological parameters of radioresistance. C. Evaluation of relevant biomarkers in clinical studies as a basis for stratification for treatment strategies. Panel B adapted from ref. 34: The Impact of Tumor Biology on Cancer Treatment and Multidisciplinary Strategies, “Tumor biology's impact on clinical cure rates,” 2009, p. 323-34, Baumann M, Krause M, with permission of Springer. CSC, cancer stem cells; TCP, tumor control probability.
Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 19
Author Manuscript Author Manuscript
Figure 0007
Author Manuscript Figure 0008
Author Manuscript
Figure 5. Illustration of utility of Genetically Engineered Mouse Models (GEMM) in the investigation of mechanism for Stereotactic Body Radiotherapy (SBRT)
A. Dual recombinase (Flp + Cre recombinases) technology can be employed to dissect the mechanism of tumor response to SBRT. Genetically engineered mice were generated so that injection of an adenovirus expressing Flp initiates primary cancer development (47). The mice also express Cre recombinase specifically from stromal cells (49). B. Deleting ATM specifically in endothelial cells sensitized tumor endothelial cells to radiation. This increased growth delay after SBRT (49) but with high-dose SBRT, which was capable of achieving
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 20
Author Manuscript Author Manuscript
local control in some mice, sensitizing endothelial cells to radiation failed to improve local control (72). Figure 5A adapted from “Generation of primary tumors with Flp recombinase in FRTflanked p53 mice” by Lee et al. (47). © 2012. Published by The Company of Biologists Ltd. This figure is licensed under a Creative Commons Attribution 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms. The article in which this figure originally appeared is published with open access at http://dmm.biologists.org/. The molecular biology icons that appear in Fig. 5B (top) are from Science Slides, VisiScience Corp., Chapel Hill, NC. The graph (left) in Fig. 5B is republished from ref. 49 with permission of the American Society for Clinical Investigation, from “ATM deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium,” Moding EJ, Lee CL, Castle KD, Oh P, Mao L, Zha S, et al., vol 124, © 2014; permission conveyed through Copyright Clearance Center, Inc. The graph (right) in Fig. 5B is from ref. 72: Moding EJ, Castle KD, Perez BA, Oh P, Min HD, Norris H, et al. Tumor cells, but not endothelial cells, mediate eradication of primary sarcomas by stereotactic body radiation therapy. Sci Transl Med; 2015;7:278ra34. Reprinted with permission from the American Association for the Advancement of Science.
Author Manuscript Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01. Published in final edited form as: Clin Cancer Res. 2016 July 1; 22(13): 3138–3147. doi:10.1158/1078-0432.CCR-16-0069.
Improving the Predictive Value of Preclinical Studies in Support of Radiotherapy Clinical Trials C. Norman Coleman1, Geoff S. Higgins2, J. Martin Brown3, Michael Baumann4, David G. Kirsch5, Henning Willers6, Pataje G.S. Prasanna1, Mark W. Dewhirst7, Eric J. Bernhard1, and Mansoor M. Ahmed1
Author Manuscript
1Radiation
Research Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, Maryland 2Cancer Research UK/ Medical Research Council, Oxford Institute for Radiation Oncology, University of Oxford, United Kingdom 3Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 4OncoRay National Center for Radiation Research, Technische Universität Dresden/ Helmholtz-Zenrtum Dresden-Rossendorf, Dresden, Germany and German Cancer Consortium, Dresden/ German Cancer Research Center (DKFZ) 5Departments of Radiation Oncology and Pharmacology and Cancer Biology, Duke University, Durham, North Carolina 6Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 7Departments of Radiation Oncology, Pathology and Biomedical Engineering, Duke University, Durham, North Carolina
Author Manuscript
Abstract
Author Manuscript
There is an urgent need to improve reproducibility and translatability of preclinical data in order to fully exploit opportunities for molecular therapeutics involving radiation and radio-chemotherapy. For in vitro the clonogenic assay remains the current state-of-the-art of preclinical assays, while newer moderate- and high-throughput assays offer the potential for rapid initial screening. Studies of radiation response modification by molecularly targeted agents can be improved using more physiologic 3D culture models. Elucidating effects on the cancer stem cells (CSC, and CSC-like) and developing biomarkers for defining targets and measuring responses are also important. In vivo studies are necessary to confirm in vitro findings, further define mechanism of action and address immune modulation and treatment-induced modification of the microenvironment. Newer in vivo models include genetically engineered and patient derived xenograft mouse models and spontaneously occurring cancers in domesticated animals. Selection of appropriate endpoints is important for in vivo studies, for example, regrowth delay measures bulk tumor killing while local tumor control assesses effects on CSC. The reliability of individual assays requires standardization of procedures and cross-laboratory validation. Radiation modifiers must be tested as part of clinical standard of care, which includes radio-chemotherapy for most tumors. Radiation models
Corresponding author: C. Norman Coleman, Radiation Research Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, 9609 Medical Center Drive, 3W102, Bethesda, MD 20892-9727. Phone: 240-276-5690; Fax: 240-276-5827; [email protected]. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Disclosure of potential conflicts of interest: No potential conflicts of interest were disclosed by the other authors.
Coleman et al.
Page 2
Author Manuscript
are compatible with, but also differ from those used for drug screening. Furthermore, the mechanism of a drug as a chemotherapy enhancer may be different than its interaction with radiation and/or radio-chemotherapy. This provides an opportunity to expand the use of moleculartargeted agents.
INTRODUCTION The era of personalized and precision medicine has emerged with a multitude of molecularly targeted drugs, immune modifiers and new classifications of cancer based on biologic/ genomic characteristics in addition to the organ of origin. Appropriate preclinical studies are critical to optimize targeted therapeutic strategies and clinical trial design to benefit the patients and also enhance the return on investment in translational research.
Author Manuscript
Approximately 60% of cancer patients in the developed world receive radiotherapy, often combined with systemic agents. Radiotherapy is a critical component of comprehensive cancer treatment in the developing world (1). It is effective and often curative, but would be more so if radiosensitizers, radioprotectors, and predictive biomarkers of patient and tumor radiation sensitivity were employed (2, 3). Radiation modifiers have been reviewed recently (4-8), as has the potential for radiation therapy to enhance the effectiveness of immunotherapeutics (9-11) to improve local tumor control as well as to induce abscopal effects that result in concomitant responses in distant metastases (12, 13).
Author Manuscript
One impediment to drug development has been irreproducibility of preclinical data (14). A 2012 NCI Radiation Research Program (RRP) workshop examined six randomized clinical trials from the Radiation Therapy Oncology Group that resulted in null outcomes (15). This result may be due in part to the quality and validity of the pre-clinical data. Stone et al. (16) after examining the details of 125 reported in vitro and in vivo preclinical studies on radiation-modifiers concluded that future preclinical studies must include: a) use of appropriate preclinical models that best represent the clinical setting of extant “standard-ofcare” multi-modality cancer therapy, b) fastidious calibration and dosimetry of radiation sources (17), c) detailed and accurate descriptions of experimental methodology and results, and d) clinically relevant drugs, doses, schedules, and assay conditions. Of critical importance is the recognition that the mechanism of action of a molecular-targeted drug may be different when used by itself to target a specific tumor pathway compared to how it affects combination therapy that includes radiation.
Author Manuscript
Here we review issues that could enhance the strength of preclinical models of radiationmodifying drugs in leading to early phase clinical trials. The perspective includes specific aspects of in vitro assay systems and in vivo rodent and companion canine tumor models with the goal of identifying critical steps, gaps and new approaches toward enhancing the use of rapidly emerging discoveries in cancer treatment with radiation therapy. Recommendations are presented with the recognition of substantial ongoing changes in cell lines and models for screening drugs for cancer treatment moving from the NCI- 60 cell lines (18) to patient derived xenografts (PDX) (19). While the radiation-modifying drugs could enhance the immune response through the radiation-induced changes in the tumor cell, this review does not include discussion of models for screening and assessing Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 3
Author Manuscript
immunotherapy. Radiation-induced immune-modulation is a major topic in itself now under active development including novel animal models with functioning humanized immune systems (20).
FOCUS AREAS: PRECLINICAL RADIOBIOLOGICAL ASSAYS
Author Manuscript
Key components of the workflow of pre-clinical radiobiological assays (Figure 1A) and potential avenues to enhance standards and quality (Figure 1B) are discussed. There is general agreement that the ability of a treated cell to form a colony, as measured by the “clonogenic assay” (21), should be considered the “gold standard” for testing radiation modifiers, although a number of commercial and short-term assays likely will be required for initial screening of drugs followed by clonogenic assays for confirmation of results wherever possible. The clonogenic assay does not work for all cell lines and this is currently problematic for patient derived in vitro models (18, 22, 23). In the future it is envisioned that short term assays using patient-derived cell lines and 3D models will be benchmarked against the gold standard clonogenic assay, using cell lines that are amenable to this endpoint. In-Vitro Radiosensitivity Screening Assays Higgins et al. (24) reported two types of screening assays for determining new radiosensitization targets including a DNA damage readout and a medium/high throughput clonogenic assay. The DNA damage readout assay (Figure 2A) was used with a siRNA library targeted against 200 DNA Damage Response (DDR) genes. Screening of both tumor and normal cell lines identified polymerase theta (POLQ) as a tumor specific radiosensitization target.
Author Manuscript
The medium/high throughput clonogenic assay (Figure 2B) uses an automated 96-well plate colony counting platform with the cell kill measurable to 2 logs (0.01) surviving fraction (25). Screening 10,000 genes in HeLa cells in 8 months identified several new targets. One of these is Thiamin pyrophosphokinase 1 (TPK1). The radiosensitizing effects of TPK1 knockdown was subsequently investigated in several other cell lines and confirmed to radiosensitize tumor but not normal cell lines. Work in progress includes evaluating suitability of other cells for this assay system, using drugs in addition to gene knockdowns and validation of hits in in vivo models.
Author Manuscript
Lin et al. (26) describe a novel High Content Clonogenic Survival Assay that is similar in concept to that of Higgins et al. (24). This assay performed well compared to traditional clonogenic assay with an upper radiation dose limit of 6 Gy (26). This assay was used in a drug screen to identify trametinib as a sensitizer for K-RAS mutant tumor cells. Like the assay of Higgins et al. (24), the range of SF is limited to 2 logs of kill, but notably, both measure cell killing rather than inhibition of proliferation as in most in short-term viability/ proliferation assays. Liu et al. reported an automated high-throughput (HTS) drug-screening platform (Figure 3) (27) based on short-term cell proliferation/survival assays (3-5 days incubation). These assays were used to derive a Short-term Radiosensitization Factor at 2 Gy (SRF2Gy), which
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 4
Author Manuscript
correlates with standard Dose Enhancement Factors (DEF) derived from clonogenic survival assays using radiation doses up to 8 Gy. The correlation between the SRF2Gy and the DEF at SF of 0.1 (DEFSF0.1) had a sensitivity and specificity of >80%. Drug-induced short-term radiosensitization was accompanied by changes in the mode of cell death such as senescence or apoptosis, thus providing a mechanistic basis as to why short-term endpoints could effectively predict radiosensitization in a clonogenic assay. The results obtained with this assay underscored the impact of genomic heterogeneity among tumors and the need to examine the activity of a targeted drug in a broad range of tumors. Further development of this automated platform would allow high-throughput initial screening of drugs against tumors from a range of established and patient-derived cell lines with defined genomic backgrounds for establishing drug-biomarker-radiosensitization relationships. In-Vitro: Endpoints and 3D Models
Author Manuscript
Short-term assay endpoints include growth inhibition (analysis of cell number after 2-4 days of exposure to an agent by assays such as MTT or XTT), cell viability analysis (trypan blue exclusion), and measures of apoptosis. The results of short-term assays do not always correlate with long-term assays that measure tumor clonogens (clonogenic assay and human tumor stem cell assays). For example, Brown and Wouters showed that while differences in genetic makeup (p53 mutation status) can change the frequency of apoptosis (a short-term assay) induced by radiation or chemotherapy (28), it plays little or no role in clonogenic survival that captures all mechanisms of cell death (29). In contrast, a positive impact of p53 mutation on increased cell sensitivity to DNA damaging agents was detected by short-term assay in the NCI-60 cell line panel (30). Thus it is important to understand the mechanism by which cancer cells die (31), and its impact on the endpoint measured in the various assays.
Author Manuscript Author Manuscript
There are significant differences between 2D and 3D cell culture systems. For example, Eke et al. showed that cells cultured in a 3D extracellular matrix are more radio- and chemoresistant compared to cells grown under 2D cell culture conditions (32). A co-culture assay platform was developed for drug screening via high-content analysis that could further mimic in vivo systems (33). Our conclusion is that clonogenic or similar long-term assays are essential tools for validating and standardizing the testing of radiation modifiers in HTS assays. While HTS is feasible with certain cells using a clonogenic survival endpoint, patient-derived tumor models will likely require non-clonogenic endpoints. 3D systems including those with extracellular matrix and with co-culture with normal cells represent approaches for initial screening of a limited number of compounds as well as for secondary screens of lead compounds identified in initial 2D screens. Validating the effects of novel radiation modifiers by examining their effects on a relevant panel of tumor and normal cell lines is key to establishing their clinical relevance. No in vitro cell culture assays will be able to fully assess the efficacy of agents that work through the microenvironment such as improved tumor oxygenation, altered immune cell infiltration or altered tumor vasculature.
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 5
In-Vivo: Approaches
Author Manuscript Author Manuscript Author Manuscript
Tumor Control Dose (TCD50) assay—Baumann and Krause (34) have reported extensively on the use of Tumor Control Dose (TCD50) assay (i.e., the radiation dose that results in a 50% probability of tumor control) as a cancer stem cell (CSC, also referred to as tumor-initiating cells or CSC-like) related endpoint in preclinical tumor models. Data from Hill and Milas (35) demonstrated the importance of CSC number and tumor control in the TCD50 assay (34) (Figure 4). They demonstrated an inverse linear correlation between their TD50 (Take Dose 50, i.e., the cell number necessary to achieve a tumor growth in 50% of the host animals) and the TCD50 after single-dose irradiation under homogeneous hypoxia (35-37). The importance of inactivation of CSC by radiotherapy for local tumor control is supported by a number of further experiments (38) and recent clinical data, for example, for patients with early stage laryngeal cancer using the putative CSC marker CD44 (36, 39), CSC number correlated with permanent local tumor control. In line with this result, a multicenter retrospective analysis of postoperative radiochemotherapy in HPV-negative high-risk HNSCC revealed a significant correlation between high levels of CD44 protein expression as well as mRNA expressions of other putative CSC markers and low locoregional tumor control after radiochemotherapy (40). A further example is the observation that low 26S proteasome activity, which leads to CSC-like phenotype with higher self-renewal capacity and higher tumorigenicity, correlates with locoregional control in HNSCC (41). Similar correlations have also been found for other tumor types investigated using a variety of putative CSC markers (42). It is important to note that so-called CSC markers do not directly indicate a single CSC, but rather correlate with enrichment of CSCs in a subpopulation. In addition, remarkable plasticity has been recognized between non-CSC and CSC phenotype in tumor cells (43, 44). For example, therapeutic interventions including fractionated irradiation may lead to epigenetic reprogramming of non-CSC to CSC-like tumor cells (45). Despite these important limitations, the clinical and preclinical observations described above indicate that surrogate markers of CSC density and number (36) are significantly correlated with the probability of local tumor control after radiotherapy, supporting the need for in vivo assays to reflect CSC inactivation.
Author Manuscript
Tumor growth delay assays may not always concur with the TCD50 assays (46). However, growth delay experiments are useful for gaining mechanistic insights and also because shrinkage and growth delay provide clinical benefit. Thus, an unresolved issue is whether there is an optimal series of in vivo assay(s) that should be done prior to proposing a clinical trial. TCD50 assays are performed much less commonly than growth delay assays, because of the larger number of animals and higher cost. Nevertheless, before clinical trials with curative endpoints are initiated, TCD50 assays, which better reflect CSC inactivation, should be considered to reduce the chance of a negative trial. This may require co-operation between specialized laboratories for practical reasons. While the TCD50 assay has been widely used in radiotherapy studies, the same arguments for their use can also be made for other therapeutic interventions when their curative potential is tested (38) Genetically engineered mouse models (GEMM)—GEMMs allow for the generation of primary tumors that develop in a mouse with an intact immune system and within a native microenvironment. Therefore, GEMMs may be particularly useful in
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 6
Author Manuscript
modeling the response of cancer to radiation and immunotherapy. Because GEMM cancers can be generated in a temporally and spatially restricted manner (47, 48), these models can be used to study the response of cancer to chemo-radiotherapy in vivo. One of the most attractive features of GEMMs is the ability to use different recombinases to mutate genes in specific cell types for mechanistic studies. Kirsch et al. (48) have used a dual recombinase GEMM to answer a fundamental question in radiobiology: Is tumor sensitivity defined by the tumor cell or host stromal components? Figure 5 outlines the results of these studies published in Moding et al. (49). With the development of Flp-regulated CreER alleles (50), dual recombinase technology can also be applied for sequential mutagenesis within tumor cells to investigate mechanisms of tumor maintenance and response to radiation therapy. These approaches could be expanded to include newer gene technologies such as CRISPR/ Cas9.
Author Manuscript
Other animal models—Companion animal studies, primarily in dogs, are a focus of the Comparative Oncology Program at the NCI. Increased interest in these models is highlighted in a recent report by the Institute of Medicine, summarizing a 2-day workshop held in 2015. A summary of this conference has recently been reported (51). These offer unique opportunities for drug and chemo-radiotherapy development (52). Canine tumors are genetically diverse, grow relatively slowly, exhibit spatio-temporal heterogeneity and diversity, are amenable to imaging studies, and can help assess cost-effective treatment choices (53). Additionally, they are not subject to HIPAA regulations, allowing for greater flexibility in conducting retrospective tissue analyses. An example of the type of data from a canine trial is provided in Supplementary Figure S1. In-Vivo: Biomarkers and Other Endpoints
Author Manuscript Author Manuscript
Other potential confounders and considerations should be taken into account when using the results of a screening assay for a combined radio-chemotherapy clinical trial. These include: a) the differential impact of the drug on the CSC and non-CSC compartments, b) the effect of the tumor microenvironment on radiation response (e.g., hypoxia) or the other components of the microenvironment as noted above, and c) the fate of various cell populations following fractionated irradiation. The impact of multi-fraction irradiation on altering the cellular molecular phenotype has been reviewed by Makinde et al. (54). Radiation-inducible targets may be exploitable for immunotherapy and molecular-targeted therapy. A comprehensive discussion of biomarkers is beyond the scope of this article, but biomarker development is an essential part of drug development and treatment selection. The γ-H2AX biomarker is frequently used for assessing the impact of therapy (55). Other DNA damage and tumor biomarkers will provide enhanced and potentially early predictive information. Opportunities exist to use tumor radiation resistance/sensitivity biomarkers to escalate/de-escalate radiation dose and to spare normal tissue toxicity. Biomarkers that assess tumor characteristics are being used for making treatment choices in clinical trials (56-58).
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 7
Author Manuscript
OTHER CONSIDERATIONS In addition to the radiobiological issues outlined above, there are additional general points to consider. Gene expression patterns can vary based on the location of the tumor cells in a tissue culture or the location of implantation in a rodent model - xenograft vs. orthotopic (59). Gene silencing is often used to study the effect of targeting a specific molecular pathway with drug(s) in an experimental model. However, John-Aryankalayil et al. showed that the gene expression pattern can be substantially different when the target gene is silenced in comparison to that seen when a pathway is drugged and also that drug concentrations in excess of what is clinically relevant can lead to mechanisms of action that may be irrelevant in the clinic (60).
Author Manuscript Author Manuscript
Spatio-temporal heterogeneity within tumors during tumor evolution, at the time of initial biopsy and profiling, and over time during and after treatment is another key and highly complex consideration. The provocative report in renal cell cancer by Gerlinger et al. demonstrated the heterogeneity in tumors present de novo and the likelihood of adaptation/ evolution during therapy (25). The complexity of heterogeneity includes genomic heterogeneity, epigenetic changes in tumor phenotype, environmental and metabolic influences on treatment outcome, normal tissue-tumor cell interaction, including cell-to-cell signaling, and immunological responses (61). In vivo organoid models (62) may mimic the hypoxic aspects of heterogeneity as the 3D models (32, 33) can reproduce cell-matrix interaction. The move to PDX addresses certain aspects of heterogeneity (63) but many important factors relating to treatment outcome as well as evolution under the treatment conditions will not be accounted for in a pre-treatment xenograft (64, 65). While speculative, given the ability to target radiation dose to specific volumes, radiotherapy may be a very useful “drug” with which to alter tumor behavior in a way that makes treatments more effective, in addition to the cell killing by radiation itself (54, 66).
TOWARD IMPROVED PRE-CLINICAL MODELS OF RADIATION MODIFIERS
Author Manuscript
A pathway for the development of radiation modifiers published recently (8, 67) suggests modifications to address the new biology; however time-honored assessments often referred to pejoratively as “classical” remain critical to both drug and radiation clinical trials (66). While animal normal tissue models may provide only limited information, normal tissue injury remains an integral part of preclinical development (67, 68) The need for studies will depend on the size of the treatment field as highly focused radiation fields in some regimens are designed to be at tissue-damaging doses to limited volumes and thereby minimizing clinically significant late effects. Critical issues raised about translation of pre-clinical study results to clinical trials are being addressed (69, 70). Recognizing the rapid evolution in tumor biology and model systems and our reluctance to be prescriptive in such a complex field, a general approach to preclinical assessment of radiation modifiers may be as follows (see Figure 1A).
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 8
Author Manuscript
Step 1. “HTS” screens need to be harmonized for 2D cultures so that comparable results can reliably obtained by different laboratories. The techniques themselves can be varied as long as the results are reasonably compatible and/or differences understood. In settings in which a limited number of compounds are being tested, the initial screening may be Step 2.
Author Manuscript
Step 2. When clonogenic assays are possible, these should be conducted as they measure cell killing down to 3-4 log kill and not just growth inhibition. Notably, few drugs are subject to a clonogenic assay and/or cell survival below 1 log is rarely assessed in short-term viability/ proliferation assay. In vitro assays should consider CSC-like cells that are relevant for radioresistance, for example through the use of tumor spheres. Further, 3D models can help approximate some microenvironmental factors. Prior to going into animal models, Step 2 may also include assessing the impact of concurrently given chemotherapeutics on radiosensitizing drug effects. The inclusion of patient-derived tumor models is recommended if the initial screen was done on established cancer cell lines. In general, Step 2 narrows down the number compounds that will undergo the next step in testing. Step 3. Assessment of efficacy in murine models should include treatment regimens that are clinically relevant. This will include combinations of radiation plus drugs that are the standard of care and an appropriate radiation schedule. While regrowth delay assays may not necessarily correspond to local tumor control, they are useful for evaluating drug-radiation interaction, mechanisms of action and microenvironmental effects. The initial models may be xenografts as are now employed or newer models from patient-derived tumors with the models selected based on their utility for predicting clinical results (16).
Author Manuscript
Step 4. At this point, expansion of in vivo systems beyond growth delay should be selectively applied based on the specific mechanistic issues being addressed and also on the aim of assessing local control as a clinically relevant endpoint, such as with the TCD50 assay, GEMMs can help determine mechanisms of action and which cells are the “target”, and companion canine models have all the components of spontaneous human tumors, as well as providing needed care to the animals. It may also be equally important to study normal tissue toxicity to critical organs in tumor bearing animal models, to determine the overall risk-benefit ratio.
Author Manuscript
Figure 1 summarizes the work flow sequence for radiation modifier preclinical development and also the hierarchy of steps to be followed with increasingly stringent laboratory protocols for preclinical radiobiology assays to ensure rigorous quality control, assure validity of pre-clinical data to conduct clinical studies and then provide broad application beyond the single laboratory that developed the assay. Such attention to detail is a key issue for the Food and Drug Administration for Laboratory Developed tests (71).
CONCLUSIONS Rapid advances in cancer biology are uncovering many potential targets for cancer treatment. This has led to a need to screen potential drugs/treatments with accuracy and efficiency so that subsequent assessments have higher chances of choosing effective
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 9
Author Manuscript
treatments. Establishing biomarkers that can be used to select the optimal treatment and monitor its effects will facilitate this process. Cancer researchers and physicians, while working to improve the knowledge in their specific fields and enhance the efficacy and reduce normal tissue toxicity need to be cognizant that the best treatment for a given patient may not necessarily be contained within their particular area of interest/research focus. To help them in treatment selection, results across preclinical studies need to be comparable, which requires fastidious assay development and crossplatform comparisons. In this context we stress the continued importance of well-done preclinical trials that are comparable between laboratories and platforms. The continued utility of “classic” methodologies in chemotherapy and radiation therapy including using clinically relevant drug doses, schedules, and assay conditions should not be overlooked in these trials.
Author Manuscript
It is expected that the relationship between the “new biology” and advancing clinical care will involve complex multi-modality care requiring knowledge of the initial tumor characteristics, biomarkers of tumor response and adaptation to treatment before treatment resistance arises. The potential for the use of radiation therapy as curative and palliative treatment (1) provides unique opportunities to exploit the technological advances in radiation together with molecular-targeted and immune-modulating agents. There is potential to use a spectrum of radiation treatments including highly focused, broad field/ whole body or systemic radiation at specified doses and dose rates, which will open up possibilities for combination treatments requiring careful development, implementation, and ongoing assessment. These are the “next steps” for novel biologically-based radiation oncology.
Author Manuscript
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments M. Baumann reports receiving a commercial research grant from Merck. D.G. Kirsch reports receiving a commercial research grant from GlaxoSmithKline. M.W. Dewhirst reports receiving commercial research grants from Aerpio Therapeutics, BioMimetix Pharmaceutical, and Janssen; has ownership interest in Celsion; and was a consultant/advisory board member for EMD Serono. Grant Support: H. Willers is supported by the American Cancer Society (123420RSG-12-224-01-DMC).
Author Manuscript
REFERENCES 1. Atun R, Jaffray DA, Barton MB, Bray F, Baumann M, Vikram B, et al. Expanding global access to radiotherapy. Lancet Oncol. 2015; 16:1153–86. [PubMed: 26419354] 2. Begg AC, Stewart FA, Vens C. Strategies to improve radiotherapy with targeted drugs. Nat Rev Cancer. 2011; 11:239–53. [PubMed: 21430696] 3. Schaue D, McBride WH. Opportunities and challenges of radiotherapy for treating cancer. Nat Rev Clin Oncol. 2015; 12:527–40. [PubMed: 26122185]
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 10
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
4. Higgins GS, O'Cathail SM, Muschel RJ, McKenna WG. Drug radiotherapy combinations: review of previous failures and reasons for future optimism. Cancer Treat Rev. 2015; 41:105–13. [PubMed: 25579753] 5. Liauw SL, Connell PP, Weichselbaum RR. New paradigms and future challenges in radiation oncology: an update of biological targets and technology. Sci Transl Med. 2013; 5:173sr2. [PubMed: 23427246] 6. Morgan MA, Parsels LA, Maybaum J, Lawrence TS. Improving the efficacy of chemoradiation with targeted agents. Cancer Discov. 2014; 4:280–91. [PubMed: 24550033] 7. Le QT, Shirato H, Giaccia AJ, Koong AC. Emerging treatment paradigms in radiation oncology. Clin Cancer Res. 2015; 21:3393–401. [PubMed: 25991820] 8. Prasanna PG, Narayanan D, Hallett K, Bernhard EJ, Ahmed MM, Evans G, et al. Radioprotectors and radiomitigators for improving radiation therapy: The Small Business Innovation Research (SBIR) gateway for accelerating clinical translation. Radiat Res. 2015; 184:235–48. [PubMed: 26284423] 9. Demaria S, Golden EB, Formenti SC. Role of local radiation therapy in cancer immunotherapy. JAMA Oncol. 2015; 1:1325–32. [PubMed: 26270858] 10. Ahmed MM, Hodge JW, Guha C, Bernhard EJ, Vikram B, Coleman CN. Harnessing the potential of radiation-induced immune modulation for cancer therapy. Cancer Immunol Res. 2013; 1:280–4. [PubMed: 24777964] 11. Ahmed MM, Guha C, Hodge JW, Jaffee E. Immunobiology of radiotherapy: new paradigms. Radiat Res. 2014; 182:123–5. [PubMed: 25036983] 12. Chandra RA, Wilhite TJ, Balboni TA, Alexander BM, Spektor A, Ott PA, et al. A systematic evaluation of abscopal responses following radiotherapy in patients with metastatic melanoma treated with ipilimumab. Oncoimmunology. 2015; 4:e1046028. [PubMed: 26451318] 13. Vanpouille-Box C, Pilones KA, Wennerberg E, Formenti SC, Demaria S. In situ vaccination by radiotherapy to improve responses to anti-CTLA-4 treatment. Vaccine. 2015; 33:7415–22. [PubMed: 26148880] 14. Challenges in Irreproducible Research [about 3 screens]. [cited 2015 Dec 15]. Available from: http://www.nature.com/news/reproducibility-1.17552 15. Liu FF, Okunieff P, Bernhard EJ, Stone HB, Yoo S, Coleman CN, et al. Lessons learned from radiation oncology clinical trials. Clin Cancer Res. 2013; 19:6089–100. [PubMed: 24043463] 16. Stone HB, Coleman CN, Deye J, Capala J, Mitchell JB, Martin JB. Preclinical data on efficacy of 10 drug-radiation combinations: evaluations, concerns and recommendations. Transl Oncol. 2016; 9:46–56. [PubMed: 26947881] 17. Desrosiers M, DeWerd L, Deye J, Lindsay P, Murphy MK, Mitch M, et al. The importance of dosimetry standardization in radiobiology. J Res Natl Inst Stand Technol. 2013; 118:403–18. [PubMed: 26401441] 18. Chabner BA. NCI-60 cell line screening: a radical departure in its time. J Natl Cancer Inst. 2016; 108:djv388. [PubMed: 26755050] 19. Ledford H. US cancer institute to overhaul tumour cell lines. Nature. 2016; 530:391. [PubMed: 26911756] 20. Sanmamed MF, Chester C, Melero I, Kohrt H. Defining the optimal murine models to investigate immune checkpoint blockers and their combination with other immunotherapies. Ann Oncol. Feb 23.2016 [Epub ahead of print]. 21. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006; 1:2315–9. [PubMed: 17406473] 22. van de Wetering M, Francies HE, Francis JM, Bounova G, Iorio F, Pronk A, et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell. 2015; 161:933–45. [PubMed: 25957691] 23. Crystal AS, Shaw AT, Sequist LV, Friboulet L, Niederst MJ, Lockerman EL, et al. Patient-derived models of acquired resistance can identify effective drug combinations for cancer. Science. 2014; 346:1480–6. [PubMed: 25394791]
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 11
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
24. Higgins GS, Prevo R, Lee YF, Helleday T, Muschel RJ, Taylor S, et al. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown. Cancer Res. 2010; 70:2984–93. [PubMed: 20233878] 25. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012; 366:883–92. [PubMed: 22397650] 26. Lin SH, Zhang J, Giri U, Stephan C, Sobieski M, Zhong L, et al. A high content clonogenic survival drug screen identifies MEK inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer. J Thorac Oncol. 2014; 9:965–73. [PubMed: 24922006] 27. Liu Q, Wang M, Kern AM, Khaled S, Han J, Yeap BY, et al. Adapting a drug screening platform to discover associations of molecular targeted radiosensitizers with genomic biomarkers. Mol Cancer Res. 2015; 13:713–20. [PubMed: 25667133] 28. Brown JM, Wouters BG. Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res. 1999; 59:1391–9. [PubMed: 10197600] 29. Brown JM, Wouters BG, Kirsch DG. Cell death identification in anticancer therapy- Letter. Cancer Res. 2015; 75:3681. [PubMed: 26286478] 30. O'Connor PM, Jackman J, Bae I, Myers TG, Fan S, Mutoh M, et al. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res. 1997; 57:4285–300. [PubMed: 9331090] 31. Rello-Varona S, Herrero-Martin D, Lopez-Alemany R, Munoz-Pinedo C, Tirado OM. "(Not) all (dead) things share the same breath": identification of cell death mechanisms in anticancer therapy. Cancer Res. 2015; 75:913–7. [PubMed: 25724677] 32. Eke I, Cordes N. Radiobiology goes 3D: how ECM and cell morphology impact on cell survival after irradiation. Radiother Oncol. 2011; 99:271–8. [PubMed: 21704412] 33. Krausz E, de Hoogt R, Gustin E, Cornelissen F, Grand-Perret T, Janssen L, et al. Translation of a tumor microenvironment mimicking 3D tumor growth co-culture assay platform to high-content screening. J Biomol Screen. 2013; 18:54–66. [PubMed: 22923784] 34. Baumann, M.; Krause, M. Tumor biology's impact on clinical cure rates. In: Molls, M.; Vaupel, P.; Nieder, C.; Anscher, MS., editors. The impact of tumor biology on cancer treatment and multidisciplinary strategies. Springer-Verlag; Berlin, Heidelberg (Germany): 2009. p. 323-34. 35. Hill RP, Milas L. The proportion of stem cells in murine tumors. Int J Radiat Oncol Biol Phys. 1989; 16:513–8. [PubMed: 2921157] 36. Baumann M, Krause M. CD44: a cancer stem cell-related biomarker with predictive potential for radiotherapy. Clin Cancer Res. 2010; 16:5091–3. [PubMed: 20861165] 37. Krause M, Yaromina A, Eicheler W, Koch U, Baumann M. Cancer stem cells: targets and potential biomarkers for radiotherapy. Clin Cancer Res. 2011; 17:7224–9. [PubMed: 21976536] 38. Baumann M, Krause M, Hill R. Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer. 2008; 8:545–54. [PubMed: 18511937] 39. de Jong MC, Pramana J, van der Wal JE, Lacko M, Peutz-Kootstra CJ, de Jong JM, et al. CD44 expression predicts local recurrence after radiotherapy in larynx cancer. Clin Cancer Res. 2010; 16:5329–38. [PubMed: 20837694] 40. Linge A, Lock S, Gudziol V, Nowak A, Lohaus F, von Neubeck C, et al. Low cancer stem cell marker expression and low hypoxia identify good prognosis subgroups in HPV(−) HNSCC after postoperative radiochemotherapy: a multicenter study of the DKTK-ROG. Clin Cancer Res. Jan 11.2016 [Epub ahead of print]. 41. Lagadec C, Vlashi E, Bhuta S, Lai C, Mischel P, Werner M, et al. Tumor cells with low proteasome subunit expression predict overall survival in head and neck cancer patients. BMC Cance. 2014; 14:152. 42. Butof R, Dubrovska A, Baumann M. Clinical perspectives of cancer stem cell research in radiation oncology. Radiother Oncol. 2013; 108:388–96. [PubMed: 23830466] 43. Kreso A, Dick JE. Evolution of the cancer stem cell model. Cell Stem Cell. 2014; 14:275–91. [PubMed: 24607403]
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 12
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
44. Vlashi E, Pajonk F. Cancer stem cells, cancer cell plasticity and radiation therapy. Semin Cancer Biol. 2015; 31:28–35. [PubMed: 25025713] 45. Cojoc M, Peitzsch C, Kurth I, Trautmann F, Kunz-Schughart LA, Telegeev GD, et al. Aldehyde dehydrogenase is regulated by beta-Catenin/TCF and promotes radioresistance in prostate cancer progenitor cells. Cancer Res. 2015; 75:1482–94. [PubMed: 25670168] 46. Budach W, Budach V, Stuschke M, Dinges S, Sack H. The TCD50 and regrowth delay assay in human tumor xenografts: differences and implications. Int J Radiat Oncol Biol Phys. 1993; 25:259–68. [PubMed: 8420873] 47. Lee CL, Moding EJ, Huang X, Li Y, Woodlief LZ, Rodrigues RC, et al. Generation of primary tumors with Flp recombinase in FRT-flanked p53 mice. Dis Model Mech. 2012; 5:397–402. [PubMed: 22228755] 48. Kirsch DG, Dinulescu DM, Miller JB, Grimm J, Santiago PM, Young NP, et al. A spatially and temporally restricted mouse model of soft tissue sarcoma. Nat Med. 2007; 13:992–7. [PubMed: 17676052] 49. Moding EJ, Lee CL, Castle KD, Oh P, Mao L, Zha S, et al. ATM deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium. J Clin Invest. 2014; 124:3325–38. [PubMed: 25036710] 50. Zhang M, Kirsch DG. The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis. Dis Model Mech. 2015; 8:1155–66. [PubMed: 26183214] 51. LeBlanc AK, Breen M, Choyke P, Dewhirst M, Fan TM, Gustafson DL, et al. Perspectives from man's best friend: National Academy of Medicine's Workshop on Comparative Oncology. Sci Transl Med. 2016; 8:324. 52. LeBlanc AK, Mazcko CN, Khanna C. Defining the value of a comparative approach to cancer drug development. Clin Cancer Res. Dec 28.2015 [Epub ahead of print]. 53. Chi JT, Thrall DE, Jiang C, Snyder S, Fels D, Landon C, et al. Comparison of genomics and functional imaging from canine sarcomas treated with thermoradiotherapy predicts therapeutic response and identifies combination therapeutics. Clin Cancer Res. 2011; 17:2549–60. [PubMed: 21292819] 54. Makinde AY, John-Aryankalayil M, Palayoor ST, Cerna D, Coleman CN. Radiation survivors: understanding and exploiting the phenotype following fractionated radiation therapy. Mol Cancer Res. 2013; 11:5–12. [PubMed: 23175523] 55. Koch U, Hohne K, von Neubeck C, Thames HD, Yaromina A, Dahm-Daphi J, et al. Residual gamma H2AX foci predict local tumour control after radiotherapy. Radiother Oncol. 2013; 108:434–9. [PubMed: 23891089] 56. Redig AJ, Janne PA. Basket trials and the evolution of clinical trial design in an era of genomic medicine. J Clin Oncol. 2015; 33:975–7. [PubMed: 25667288] 57. NCI-Molecular Analysis for Therapy Choice (NCI-MATCH) Trial [about 9 screens]. [cited 2015 Dec 31]. Available from: http://www.cancer.gov/about-cancer/treatment/clinical-trials/ncisupported/nci-match 58. Baumann M, Krause M, Overgaard J, Debus J, Bentzen SM, Daartz J, et al. Radiation oncology in the era of precision medicine. Nat Rev Cancer. 2016; 16:234–49. [PubMed: 27009394] 59. Kahn J, Tofilon PJ, Camphausen K. Preclinical models in radiation oncology. Radiat Oncol. 2012; 7:223. [PubMed: 23270380] 60. John-Aryankalayil M, Palayoor ST, Cerna D, Falduto MT, Magnuson SR, Coleman CN. NS-398, ibuprofen, and cyclooxygenase-2 RNA interference produce significantly different gene expression profiles in prostate cancer cells. Mol Cancer Ther. 2009; 8:261–73. [PubMed: 19139136] 61. Alizadeh AA, Aranda V, Bardelli A, Blanpain C, Bock C, Borowski C, et al. Toward understanding and exploiting tumor heterogeneity. Nat Med. 2015; 21:846–53. [PubMed: 26248267] 62. Hubert CG, Rivera M, Spangler LC, Wu Q, Mack SC, Prager BC, et al. A three-dimensional organoid culture system derived from human glioblastomas recapitulates the hypoxic gradients and cancer stem cell heterogeneity of tumors found in vivo. Cancer Res. Feb 19.2016 [Epub ahead of print].
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 13
Author Manuscript Author Manuscript
63. Cassidy JW, Caldas C, Bruna A. Maintaining tumor heterogeneity in patient-derived tumor xenografts. Cancer Res. 2015; 75:2963–8. [PubMed: 26180079] 64. Siolas D, Hannon GJ. Patient-derived tumor xenografts: transforming clinical samples into mouse models. Cancer Res. 2013; 73:5315–9. [PubMed: 23733750] 65. Gao H, Korn JM, Ferretti S, Monahan JE, Wang Y, Singh M, et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nat Med. 2015; 21:1318–25. [PubMed: 26479923] 66. Coleman CN, Lawrence TS, Kirsch DG. Enhancing the efficacy of radiation therapy: premises, promises, and practicality. J Clin Oncol. 2014; 32:2832–5. [PubMed: 25113766] 67. Lawrence YR, Vikram B, Dignam JJ, Chakravarti A, Machtay M, Freidlin B, et al. NCI-RTOG translational program strategic guidelines for the early-stage development of radiosensitizers. J Natl Cancer Inst. 2013; 105:11–24. [PubMed: 23231975] 68. Harrington KJ, Billingham LJ, Brunner TB, Burnet NG, Chan CS, Hoskin P, et al. Guidelines for preclinical and early phase clinical assessment of novel radiosensitisers. Br J Cancer. 2011; 105:628–39. [PubMed: 21772330] 69. Landis SC, Amara SG, Asadullah K, Austin CP, Blumenstein R, Bradley EW, et al. A call for transparent reporting to optimize the predictive value of preclinical research. Nature. 2012; 490:187–91. [PubMed: 23060188] 70. Funding Opportunity Announcement: Cooperative Agreement to Develop Targeted Agents for Use with Systemic Agents Plus Radiotherapy (U01) [about 19 screens]. [cited 2016 Mar 7]. Available from: http://grants.nih.gov/grants/guide/pa-files/PAR-16-111.html 71. Laboratory Developed Tests [about 4 screens]. [cited 2015 Dec 15]. Available from: http:// www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ ucm407296.htm 72. Moding EJ, Castle KD, Perez BA, Oh P, Min HD, Norris H, et al. Tumor cells, but not endothelial cells, mediate eradication of primary sarcomas by stereotactic body radiation therapy. Sci Transl Med. 2015; 7:278.
Author Manuscript Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 14
Author Manuscript Author Manuscript
Figure 0001
Author Manuscript Author Manuscript
Figure 0002 Figure 1. A. Workflow of pre-clinical radiobiology experimental tasks from the discovery phase to clinical trials
The critical steps are the clonogenic assay and regrowth delay assay using clinically relevant model that includes the extant standard of care that may include systemic agents. Initial drug screening may be high/moderate throughput automated assays or more targeted clonogenic screening. The options for in vivo assays include mouse xenograft models and patient derived assays. Additional studies may be needed to address mechanism of action, normal Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 15
Author Manuscript
tissue toxicity, target cells and microenvironment, such as TCD50 using GEMMs, or canine companion models. B. Steps for assay development to improve quality control and quality assurance for the preclinical radiobiology assays. Developing and standardizing assay procedures are critical for the broad clinical applicability of the assays beyond the research laboratories in which the concept is initially discovered and developed. GEMM, genetically engineered mouse models; PDX, patient derived xenograft; TCD50, Tumor control dose50.
Author Manuscript Author Manuscript Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 16
Author Manuscript Author Manuscript
Figure 0003
Author Manuscript Figure 0004
Author Manuscript
Figure 2. Moderate-/high throughput screening radiosensitivity assay and protocol
A. High throughput screening (HTS) for new radiation sensitizer targets. Cells are transfected in quadruplicate with half the plates receiving 4-Gy radiation. At 24 h plates are stained for γH2AX and read with a reader. B. Medium throughput clonogenic assay. NT, nontargeting.
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 17
Author Manuscript Author Manuscript
Figure 3. The automated drug discovery platform at the Massachusetts General Hospital Cancer Center (MGHCC)
Illustration of an automated robotic drug-screening platform to discover associations of molecular targeted radiosensitizers with genomic biomarkers. Adapted from Liu and colleagues (27). GI, gastrointestinal; GU, genitourinary; HNSCC, head and neck small cell cancer; SRF, short-term radiosensitization factor.
Author Manuscript Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 18
Author Manuscript Author Manuscript
Figure 4. Tumor control dose (TCD50) assay
Author Manuscript
A. Local tumor control depends on cancer stem cell inactivation and should therefore have an important role in preclinical in vivo assays. B. The response to radiotherapy of tumors of the same entity and size differs due to intertumoral heterogeneity in (radio)biological parameters of radioresistance. C. Evaluation of relevant biomarkers in clinical studies as a basis for stratification for treatment strategies. Panel B adapted from ref. 34: The Impact of Tumor Biology on Cancer Treatment and Multidisciplinary Strategies, “Tumor biology's impact on clinical cure rates,” 2009, p. 323-34, Baumann M, Krause M, with permission of Springer. CSC, cancer stem cells; TCP, tumor control probability.
Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 19
Author Manuscript Author Manuscript
Figure 0007
Author Manuscript Figure 0008
Author Manuscript
Figure 5. Illustration of utility of Genetically Engineered Mouse Models (GEMM) in the investigation of mechanism for Stereotactic Body Radiotherapy (SBRT)
A. Dual recombinase (Flp + Cre recombinases) technology can be employed to dissect the mechanism of tumor response to SBRT. Genetically engineered mice were generated so that injection of an adenovirus expressing Flp initiates primary cancer development (47). The mice also express Cre recombinase specifically from stromal cells (49). B. Deleting ATM specifically in endothelial cells sensitized tumor endothelial cells to radiation. This increased growth delay after SBRT (49) but with high-dose SBRT, which was capable of achieving
Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
Coleman et al.
Page 20
Author Manuscript Author Manuscript
local control in some mice, sensitizing endothelial cells to radiation failed to improve local control (72). Figure 5A adapted from “Generation of primary tumors with Flp recombinase in FRTflanked p53 mice” by Lee et al. (47). © 2012. Published by The Company of Biologists Ltd. This figure is licensed under a Creative Commons Attribution 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms. The article in which this figure originally appeared is published with open access at http://dmm.biologists.org/. The molecular biology icons that appear in Fig. 5B (top) are from Science Slides, VisiScience Corp., Chapel Hill, NC. The graph (left) in Fig. 5B is republished from ref. 49 with permission of the American Society for Clinical Investigation, from “ATM deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium,” Moding EJ, Lee CL, Castle KD, Oh P, Mao L, Zha S, et al., vol 124, © 2014; permission conveyed through Copyright Clearance Center, Inc. The graph (right) in Fig. 5B is from ref. 72: Moding EJ, Castle KD, Perez BA, Oh P, Min HD, Norris H, et al. Tumor cells, but not endothelial cells, mediate eradication of primary sarcomas by stereotactic body radiation therapy. Sci Transl Med; 2015;7:278ra34. Reprinted with permission from the American Association for the Advancement of Science.
Author Manuscript Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2017 January 01.
