I. What we sought to accomplish II. How the Think Tanks were organized and run III. Overall recommendations IV. Concluding remarks Appendix A

Tumor Immunology Think Tank

Executive Summary Introduction Recommendations

Tumor Microenvironment Think Tank

Executive Summary Introduction Recommendations

Tumor Stem Cell & Self-Renewal Genes Think Tank

Executive Summary Introduction Recommendations

Cell Decisions in Response to DNA Damage: Survival vs. Programmed Cell Death Think Tank

Executive Summary Introduction Recommendations

Cancer Etiology: Role of Exogenous and Endogenous Chemicals Think Tank

Executive Summary Introduction Recommendations

Epigenetic Mechanisms in Cancer Think Tank

Executive Summary Introduction Recommendations

Inflammation and Cancer Think Tank

Executive Summary Introduction Recommendations

Cancer Susceptibility and Resistance Think Tank

Executive Summary Introduction Recommendations

Rosters

Think Tank Participant Rosters      Tumor Immunology Think Tank      Tumor Microenvironment Think Tank      Tumor Stem Cell & Self-Renewal Genes Think Tank      Cell Decisions in Response to DNA Damage: Survival vs. Programmed Cell Death Think Tank      Cancer Etiology: Role of Exogenous and Endogenous Chemicals Think Tank      Epigenetic Mechanisms in Cancer Think Tank      Inflammation and Cancer Think Tank      Cancer Susceptibility and Resistance Think Tank Division of Cancer Biology Scientific Staff Acknowledgements

Summary Report

I. What we sought to accomplish

A year ago (in 2003), Dr. von Eschenbach charged the Division of Cancer Biology (DCB) with conducting a series of Think Tanks to assess the state of cancer biology research and to recommend to the NCI a research agenda that would accelerate progress in cancer research. The process of fulfilling this charge began with an internal identification of scientific areas of unusual promise for rapid progress. Eight areas were chosen as the topics of Think Tanks. The areas were quite different in scope and in the maturity of the scientific disciplines involved. As a result, each Think Tank had to be structured differently, to deal with the unique questions and opportunities in each area. The eight Think Tanks are listed below, each with a brief description of its goal:

1) Tumor Immunology - This area of basic cancer biology has gone from relative obscurity to intellectual vitality over the past several years, as a result of critical basic science and translational advances, coupled with new concepts and methods in basic immunology. Immunotherapy of cancer had disputed translational potential for many years, but is now an established and expanding therapeutic modality. The field has untapped promise, but basic science advances have made clear how much still needs to be known about how the immune system and tumors affect one another. In addition, clinical advances depend on improving the rate at which immunological and other biological therapies are brought to the point where they can be used in clinical studies, and on devising clinical trials structures that accurately and efficiently allow evaluation of efficacy. These basic and translational issues were the focus of discussion.

2) Tumor Microenvironment - The microenvironment in which a tumor arises has a profound influence on its progression. Tumor cells and stromal elements such as endothelial cells, fibroblasts, lymphoid and myeloid cells, extracellular matrix and cytokines interact dynamically and depend on one another for growth and survival. The tumor microenvironment has been the focus of intense interest within NCI and the scientific community for some time because it is the key to understanding tumor biology and strongly affects the likelihood of successful cancer therapy. While a consensus has been reached that this area deserves substantial additional support, this Think Tank had the goal of specifying the type of initiative(s) that would best facilitate rapid advances in knowledge about the microenvironment.

3) Tumor Stem Cell & Self-Renewal Genes - Recent results suggesting that at least some tumors contain only a small number of cells with tumor-initiating potential (loosely termed tumor stem cells), have profound implications for carcinogenesis, cancer biology and cancer therapy. Think Tank participants discussed current evidence for the existence of tumor stem cells in different tumor types, how they might arise, and the genetic (and possibly epigenetic) pathways important in maintaining the tumor stem cell state.

4) Cell Decisions in Response to DNA Damage: Survival vs. Programmed Cell Death - When cells encounter DNA damage, they can repair the damage or decide to try to live with it, either of which leads to survival, or they can choose to undergo programmed cell death. This life or death decision involves a complex interplay between DNA damage sensing and repair pathways, the pathways controlling the cell cycle, and cell death mechanisms. With an understanding of how these decisions are made, the response to radiation and/or chemotherapy could be improved by increasing the likelihood that cancer cells would die in response to therapy-induced damage while decreasing the chance that normal cells would die. Experts in DNA damage, cell cycle control, and apoptosis, who do not meet regularly, met at this Think Tank to consider prospects for fruitful interaction.

5) Cancer Etiology: Role of Exogenous and Endogenous Chemicals - Classical studies in chemical carcinogenesis have definitively established an important role for exogenous and endogenous chemicals in cancer development and defined some of the mechanisms involved, particularly those related to DNA adduct formation. Other areas, such as the role of damage to proteins and lipids, and identifying markers of exposure, have received less emphasis. The challenge in the field is to build on what has been done by integrating with other fields of biology (particularly integrative cancer biology and cancer susceptibility) to build a system-wide understanding of the complex process of carcinogenesis. The participants discussed the connections that exist between chemical carcinogenesis and areas such as inflammation, biological carcinogenesis, reactive oxygen species and systems biology, and how to strengthen them.

6) Epigenetic Mechanisms in Cancer - Epigenetic mechanisms are those that lead to heritable changes in cell behavior without changes in DNA sequence. The dramatic changes in gene expression that characterize tumor cells come about through both genetic and epigenetic mechanisms. Dramatic advances in genetics and genomics, and smaller advances in epigenetics, have sharpened our appreciation of the critical role that epigenetics plays in development and cancer, through DNA methylation, histone modification and changes in higher order chromatin structure. The Think Tank was organized to discuss evidence for epigenetic phenomena important in cancer and to prioritize NCI activities to advance the field.

7) Inflammation and Cancer - Chronic inflammatory disease has been known for many years to predispose to cancer development, and most tumor sites show evidence of ongoing inflammation. Inflammation can benefit a tumor by leading to production of mutagens, such as reactive oxygen and nitrogen species, and growth-stimulatory cytokines. At the same time, the proper inflammatory signals are necessary to allow the development of an effective immune response against the tumor. The participants in the Think Tank discussed how to sort out the positive and negative influences of inflammation in cancer, including that due to infectious agents, and how this understanding is expected to contribute to more effective strategies for cancer prevention and treatment.

8) Cancer Susceptibility and Resistance - The consensus of two prior meetings of mouse geneticists, population scientists, and statisticians is that the ability to understand human cancers as complex traits will require new statistical and computational methods for modeling gene/environment interactions, and assembling data for high-level gene network analysis. This Think Tank was set up to explore novel developments in mathematics and engineering design and their application to cancer susceptibility research, both in modeling human populations and hypothesis testing/candidate gene validation in rodent models.

9) Integrative Cancer Biology - The first Think Tank area to be addressed with a major initiative was systems biology or, in this specific context, integrative cancer biology. Cancer is sufficiently complex that it will never be adequately understood based on the expression of a few genes or the regulation of a few signal transduction pathways in the transformed cell. It is an emergent property of derangements in complex intracellular systems in a context set by equally complex interactions of the tumor cell with intercellular environment. No report is included here for the Integrative Cancer Biology Think Tank because a solicitation has already been done for Integrative Cancer Biology Programs, and these have now been funded. This was the key recommendation of the Think Tank. Recommendations from several of the other Think Tanks dealt with the need to foster this area (see below for discussion), so this is likely to remain a topic of great interest for NCI for the foreseeable future.

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II. How the Think Tanks were organized and run

The general goal of each Think Tank was to assess a specific area of cancer biology, determining where the science is and where it is going, and asking what NCI could or should do to facilitate progress. In areas that had been assessed previously in workshops and where NCI had made a commitment to do initiatives, such as the tumor microenvironment, the emphasis was entirely on what NCI should do. In other areas, where less had been done and no funding commitment existed, the emphasis was more on assessing the state of science in the field. A general charge was developed for each Think Tank, as follows:

Questions to Consider:

• Where does the field stand today?


• What would we have to know to move the field forward dramatically?


• What are the gaps and/or roadblocks to progress?


• Are there areas of expertise that need to be, but have not been, brought to bear on the problems in this field? If so, how can this be remedied?


• What cross-cutting tool, enabling technology and/or infrastructure needs to be developed?


• What can NCI do to move the field forward?

Each Think Tank was unique in scope and maturity of the field, so each had subtly different goals and had to be organized somewhat differently. Accordingly, the organizers of each Think Tank were given guidelines or "points to consider," rather than firm rules. These organizational guidelines are included as Appendix A. A critical element of every Think Tank was the selection of outside co-chairs. These individuals are distinguished scientists with unusual breadth of vision in the area to be discussed and strong organizational ability. They were recruited very early in the planning process, and played a leading role in identifying other participants, defining the critical questions to be discussed and setting the agenda. They were also involved in assembling the reports included here, and generally took the lead if an account of the Think Tank was prepared for publication in an appropriate scientific journal. In every case, the recommendations included in the Think Tank reports are those of the co-chairs and the other participants.

Each Think Tank was organized independently, but it was obvious from the outset that some of the Think Tanks overlapped. For example, tumor immune responses are influenced by the tumor microenvironment and cancer etiology involves a strong element of inflammation, at least as a cofactor. This was seen as an asset because it made it possible to build in a level of continuity in these areas by inviting key participants to attend two or more Think Tanks. In some cases, formal participation in two Think Tanks was not possible, but advice was always sought where overlaps were anticipated to ensure that similar issues were addressed.

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III. Overall recommendations

Each Think Tank was unique in focus and participants, and each resulted in a series of recommendations that will be addressed individually. All of the reports are appended, and for easy access the recommendations have been collected at the end of each report. This section will deal with a number of scientific and support-mechanism issues that emerged independently in multiple Think Tanks, with details on each given below. The scientific issues of the tumor microenvironment and integrative cancer biology (systems biology) were mentioned in virtually every Think Tank. Both of these subjects have been identified as high-priority areas for NCI support. The Think Tank reports provide a wealth of details on what needs to be done in these areas and reinforces their importance for cancer biology in the future. It was emphasized in these areas and others that studying cancer is impossible without also studying normal biology. The derangements in cancer can only be understood in terms of their deviations from normal behavior. This increases the scope of what must be done, and challenges us to coordinate with agencies supporting studies of normal biological development and other diseases.

Among the issues dealing with support mechanisms, it was not a surprise that the participants uniformly identified a need for additional and more flexible mechanisms to support multi-disciplinary, generally multi-institutional efforts to address complex issues like integrative cancer biology and the tumor microenvironment. A related concern was the difficulty of establishing training programs that prepare students and investigators for research in a multi-disciplinary environment. To balance this concern, each Think Tank also emphasized the indispensable contribution by, and the continuing importance of, research supported by investigator-initiated R01 grants.

Some of the issues were unexpected. Inflammation was recognized as important enough to deserve its own Think Tank, but it was surprising how prominent a role it occupied in other Think Tanks. This suggests that it needs an even more prominent role than had been envisioned in initiatives to study the tumor microenvironment, where inflammation is a nearly constant finding. A related common theme was the role of microbial flora as a cofactor in tumor development. While biological carcinogenesis, with an emphasis on cancer caused by viruses, has always been supported within NCI, examination of a cofactor role for microbes that are not directly transforming has lagged behind. This is one of several areas that bridge cancer biology and etiology. Support for technology development, in general, appears to remain a challenge despite the addition of many new programs in recent years. Think Tank participants consistently reported limitations in funds for reagent preparation (e.g., monoclonal antibodies), model development (both genetically engineered animals and complex, three-dimensional tissue cultures), and state-of-the-art imaging. Funding for critical resources needs to be factored into plans in many areas, but it was also surprising that in some instances Think Tank participants recommended that NCI make available resources, including reagents, databases, animals and facilities, that already exist. This indicates that more effort needs to be put into ensuring that all members of the cancer research community are aware of the resources NCI currently provides. If there are problems of quality or access with existing resources, these need to be evaluated as well.

The Tumor Microenvironment

Evidence continues to accumulate that growth and migration of normal epithelial cells are subject to many levels of regulation by neighboring cells, extracellular matrix, and local levels of soluble signaling molecules. Cancer cells lose critical aspects of these controls, but they lose them gradually and rarely lose them all. Thus, one way of looking at cancer initiation and progression is as an iterative and progressive renegotiation of constraints carried out between a developing clone of epithelial cancer cells and its stromal microenvironment. This perspective suggests two principal lessons. First, attempts to understand tumor behavior or to treat cancers must take into account far more than the intrinsic properties of the malignant cells to be successful. And second, attempts to model tumor behavior must go beyond using tumor cell lines cultured on plastic surfaces, to three-dimensional culture systems and in vivo studies. The Tumor Microenvironment Think Tank provided a detailed blueprint for integrated studies, and several of the other Think Tanks emphasized specific aspects of the microenvironment that are often overlooked in overviews of the subject.

1) Characterize all of the cellular and non-cellular components of the normal, wounded, and tumor tissue microenvironments. The microenvironment that sustains and shapes tumor stem cells would also be of interest. Characterization would involve probing the genomics and proteomics of stromal and tumor cells, and developing antibodies and other reagents useful for visualizing, quantitating and comparing different microenvironments, and storing the data in public databases. Static characterization would rapidly be extended to studies of dynamic interactions, using real-time imaging methods.

2) Facilitate communication and data exchange through caBIG.

3) Create and make available to the research community three-dimensional tissue culture and animal models in which microenvironmental influences can be studied.

4) Delineate the role of the microenvironment in tumor progression and metastasis, and in response to radiation and/or chemotherapy, including characterization of the metastatic tumor microenvironment.

5) Determine the role of inflammation in shaping the tumor microenvironment in the earliest phases of tumor initiation and during progression. Delineate how inflammatory processes facilitate or inhibit the development of an effective antitumor immune response. It will be critical to determine the role of the microbial flora in establishing the nature and degree of inflammation.

6) Understand the effects of androgens and/or estrogens on inflammation and other aspects of the microenvironment.

7) Explore the role of host genetics in influencing stromal elements, and determine whether mutations or epigenetic changes in the stroma influence tumor growth and capacity to metastasize.

8) Encourage the use of state-of-the-art imaging technologies in microenvironment studies and the development of techniques to bypass current limits on imaging. This is critical because only imaging has the capacity to capture the dynamic and contact-mediated aspects of this complex system.

9) Provide an environment conducive to interdisciplinary training programs.

10) Translate the basic knowledge obtained to improve diagnosis and early detection of cancer, and to discover and validate therapeutic targets derived from the tumor microenvironment.

Think Tanks other than the one focused specifically on the tumor microenvironment have influenced our sense of priorities in this area. Tumor Immunology emphasized the critical roles of the tumor and lymph node microenvironments in determining the effectiveness of antitumor immune responses. The Tumor Stem Cell Think Tank noted that the long-term proliferation potential of a tumor can controlled by a small number of tumor stem cells. Hence, the niche that supports those stem cells must be characterized and stem cell markers developed so that the most important subset of interactions can be studied. The Inflammation Think Tank highlighted evidence that inflammation is strongly associated with tumor initiation and progression, suggesting that the common inflammatory aspects of the microenvironment facilitate tumor development. At the same time, there is the paradox that inflammation, at least in infectious diseases, is associated with brisk immune responses, but tumors generally exhibit evidence of ongoing inflammation while suppressing immune responses. It is clearly insufficient to say that inflammation is present or absent; the phenomenon must be dissected to its molecular roots if interventions are to be developed. In Cancer Etiology, the macroenvironmental influences of both chemicals and microbes were discussed. Chemical exposures have impact far beyond mutagenesis, extending into a range of effects on the microenvironment. Striking evidence also exists in some tumor types that the normal microbial flora strongly influences the microenvironment. Viruses and bacteria must be assessed as important components of, or cofactors for a permissive tumor microenvironment. Finally, it was emphasized in Cancer Susceptibility that some of the complexity that has made it difficult to identify the genes responsible for individual variation in susceptibility comes from the context dependence of gene expression. Individuals may have a series of genetic polymorphisms that would predispose to cancer development, but it may not make a difference unless the microenvironment in which the tumor must develop supports the expression of the susceptibility gene(s).

Integrative Cancer Biology

As Fiscal Year 2004 is ending, NCI is funding a series of Integrative Cancer Biology Programs, the first organized foray into systems biology in the context of cancer. The Think Tanks provided strong evidence that this is an important direction for the Institute to pursue and that its influence will be felt throughout cancer research. Integrative Cancer Biology is complementary to the reductionist studies that comprise the majority of the grant portfolio. It attempts to address the complexity of many interactive and interdependent biological processes, starting by making high-throughput measurements of critical parameters. Biological lessons will be extracted from the masses of data through the use of advanced bioinformatics tools and the construction of predictive computational models of the cancer process. Although integrative biology is most often identified with the analysis of signal transduction pathways and other regulatory circuits within a single cell, it is equally applicable to complex processes involving multiple cells and extracellular molecules. Thus, the tumor microenvironment can be fully characterized only through the use of high-throughput analytical methods, and participants in the Think Tank acknowledged that a predictive model of the interactions that drive the microenvironment can be obtained only through the methods of integrative biology. The same is true of the related fields of tumor immunology and inflammation.

Other areas explored in the Think Tank process are similarly dependent on the emerging field of integrative cancer biology. Epigenetic influences on gene expression are mediated through DNA methylation, covalent modifications of histones, and higher order chromatin structure effects. Progress in the field is absolutely dependent on being able to measure these molecular changes on a genome-wide scale and knowing how to extract meaningful patterns from this mass of data. The DNA damage response and the cell cycle and cell death machinery are complex, interacting systems each made up of many quasi-stable molecular complexes. The dynamics and interactions of these systems must be understood in detail if they are to be manipulated for patient benefit in cancer. Determining how tumor stem cells function and how they come about involves achieving a molecular understanding of the cell regulatory pathways that underlie "stemness," or the ability of some cells to maintain unlimited replication potential and to divide asymmetrically into another stem cell plus a daughter cell committed to differentiation. The stem-cell genetic program is in turn heavily influenced by the cell and molecular milieu of the stem-cell niche. Etiology and susceptibility are two different views of the earliest stages of tumor development. They have been recognized for some time to involve numerous genetic and environmental influences impinging on complex cellular homeostatic mechanisms. These disciplines all need high-throughput data, bioinformatics support, and computational modeling to address critical issues.

The Challenge of Comparing the Normal and the Tumor State

The National Cancer Institute has finite resources and cancer biology presents an enormous array of promising areas of investigation directly relevant to the NCI mission. To maximize the impact of our efforts, it is tempting to focus exclusively on the cancer state, leaving studies of the normal state to other funding agencies. In six of the Think Tanks, participants explicitly recommended against this course of action, pointing out a need to understand cancer in the context of normal biology. In the Tumor Microenvironment, normal constraints on cell growth and mobility are gradually loosened as the tumor develops. We need to know much more about the normal constraints individually, and about how they are coordinated at a systems level, before the tumor microenvironment can be fully characterized. The Cell Decisions in Response to DNA Damage are similarly complex and also must be better described in the normal case before they can be manipulated for therapeutic benefit in cancer. In Tumor Immunology, the major advances in understanding that have occurred in the last ten years have come from conceptual advances in immunology as a whole. The critical questions that remain are the same for basic immunology, autoimmunity, chronic infectious diseases and cancer, although the perspectives on the questions differ slightly among these fields. Inflammation in cancer has a marked stimulatory effect on cancer growth not because of its intensity, but because it fails to resolve the way acute, physiological inflammation does. It shares this characteristic with autoimmunity and certain chronic infections, and it is from a comparison of all these states that further understanding of the process, and the ability to modulate it selectively, will come. Cancer appears to use the stem-cell program of several tissues to further its own causes, but so little is known about the regulatory program within the normal tissue stem cell and the cell-cell interactions of the stem-cell niche that it is difficult to characterize cancer stem cells or to determine the path by which they became transformed. Epigenetics is similarly a young field, in which a great deal of basic knowledge must be accumulated before its role in cancer can be clarified.

The challenge is to identify those elements of these fields that the NCI should attack with its own resources and those where it should work in coordination with other NIH Institutes and other funding agencies. Leveraging of resources is difficult, but necessary. Some of the NIH Roadmap areas are relevant to scientific issues listed above, but a great deal more remains undone. There is no large-scale project on epigenetics on the horizon, despite its documented importance in many human diseases. Similarly, while the NIH has some coordinated activities related to human embryonic stem cells, tissue-specific stem cells (with the exception perhaps of hematopoietic stem cells) have received scant attention. The Think Tank recommendations make it clear that catalyzing larger-scale studies of some critical cross-cutting biological issues must be a high priority for NCI to provide the necessary context for progress against cancer.

Mechanisms to Foster Collaborative, Interdisciplinary Research

NIH grants, built around the R01 traditional research grant, have been the engine of creativity that has brought us to the current exciting point in cancer research. The Think Tank participants uniformly acknowledged the past and continuing importance of these individual grants. During the Think Tanks, however, they focused on needs that are difficult or impossible to meet through this mechanism. These were generally large-scale efforts, especially those that required input from scientists in diverse disciplines. The Tumor Microenvironment Network, described above, is an example of the recommendations, but similar networks were suggested in immunotherapy, stem-cell research, epigenetics, etiology and susceptibility. In other cases, less formal (and smaller scale) resources for collaboration were recommended. The Inflammation Think Tank recommendations included one to "sponsor interactive fora that interface experts drawn from different disciplines to address the multi-faceted topic of inflammation and cancer at a deeper level." The Cancer Etiology Think Tank made several recommendations for collaborative undertakings, including one for instrument development and use. The Epigenetics Think Tank recommended formation of a working group to discuss and begin outlining a Human Epigenome Project. In this case, the smaller initiative could lead to the development of a large-scale effort.

For very large projects, such as a Tumor Microenvironment Network, the size and expense of the proposal makes it appropriate for funding through the RFA process. The NCI has available a wide variety of funding mechanisms and governance models for large projects of this type. It is noteworthy that in recent years RFAs at NCI and elsewhere at NIH have increasingly emphasized collaboration and interdisciplinary teamwork, part of a well publicized trend toward "team science." The large projects included in the Think Tank recommendations, including the Tumor Microenvironment Network, exemplify that trend. These recommendations came from the outside Think Tank participants, rather than NCI staff, indicating that the trends in RFAs reflect the current thinking of the research community.

Many of the recommendations were modest in cost and involved more coordination than direct research support. These recommendations were made because there are very few investigator-initiated NIH funding mechanisms that can support any of these varied activities. Critical problems in cancer research and other areas of biomedicine increasingly require a variety of expertise and/or the sharing of data or reagents in a manner that is not facilitated or sometimes even possible when support comes exclusively from grants to individual principal investigators. Constraints on collaborative and interdisciplinary research also exist at research institutions. Rigid departmental structure, intellectual property policies and concerns about indirect costs can make some types of research more difficult.

With sufficient resources, NCI could address all of these recommendations through available mechanisms such as contracts, supplements and workshops. DCB fully intends to address as many of the high-priority recommendations as possible through such efforts. What this will not do, however, is alter the fact that few such efforts can be initiated directly by the research community through investigator-initiated mechanisms. Some collaborative research can be carried out through Program Projects, but these offer a limited range of flexibility. They typically have a small number of projects, all of which normally extend for the entire project period. There is also a perception that multi-institutional Program Projects are difficult to get funded. SPORE-like grants were recommended in a couple of cases, based on the added flexibility of a changing cast of projects and integral training, but these are possible only in response to an initiative. The DCB Activities to Promote Research Collaborations (APRC) program is relatively flexible, but it is relatively small and short-term in nature. What is needed is a highly flexible, permanent program open to investigator-initiated applications to support modest-scale, collaborative, interdisciplinary research efforts. DCB will work toward the design of such a program.

Collaborative and Interdisciplinary Training

Each recommendation for a collaborative and/or interdisciplinary research program was accompanied by a recommendation for a program that would train students, postdoctoral fellows and established investigators to take optimum advantage of the opportunities such a program would create. There are differences in opinion as to the ideal way to attract and train scientists comfortable with both wet-lab biology and computational modeling, for example, or with both chemistry and biology, but there is universal agreement that any barriers that exist between disciplines need to be removed. Some barriers exist at the level of research institutions, but NCI must strive to create incentives for flexible training programs that will meet the needs for the next generation of research scientists. Three types of suggestions about training were made during the Think Tanks. One was to incorporate training into large-scale interdisciplinary initiatives. This was done in the Integrative Cancer Biology Programs, and is a feature of some other NCI programs. The second was to place some leverage back in the hands of graduate students by inaugurating individual pre-doctoral fellowships in which the range of subdisciplines and the mentor(s) could be determined by the graduate fellow and not the institution. The third was to reserve a portion of NCI postdoctoral training grants for explicitly interdisciplinary programs. While institutions are moving to respond to the need to change training paradigms, the Think Tank process made it clear that NCI must work to facilitate and accelerate such change.

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IV. Concluding Remarks

The Think Tank process provided an opportunity for both NCI funded investigators and DCB scientific staff to think broadly and deeply about the directions cancer biology must take in support of the goals of the NCI.

The individual Think Tank reports are attached. Each of the individual recommendations deserves, and will receive, consideration by DCB staff. The resulting initiatives, both large and small, will reflect a unified agenda for cancer biology over the next several years.

The cross-cutting themes, summarized here, all derive from the need and emerging ability to study cancer biology as a complex system. Integrative cancer biology will be the necessary new fundamental discipline driving this transformation. We believe that understanding the tumor microenvironment is an initiative that can focus the new tools and approaches and provide results with dramatic translational impact. To support this endeavor and others, the emerging trend toward team science as a component of cancer biology research needs to be reinforced with a reengineering of NCI funding mechanisms and approaches to encourage more collaborative and interdisciplinary interactions, accompanied by continued support of an investigator-initiated research portfolio.

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Appendix A

Guidelines for Organization of Think Tanks

1. Think Tank Goals - Concrete goals should be set early in the planning process, and modified as necessary. The goals should be communicated to the participants before the Think Tank.

2. General Structure of Agenda - Think Tanks need to be distinguished from scientific workshops. Presentations, if any, should be short and not involve primary data. Slide talks should be banned or held to a strict minimum. The bulk of the time should be devoted to brainstorming and structured group discussion.

3. Number and Type of Participants - To optimize discussion, the number of outside participants should generally be limited to fifteen. The participants should cover the critical subdisciplines and points of view. It is desirable to have a mix of established authorities and rising stars. It may also be useful to have a few key individuals who have useful perspectives but are not identified with the field in question. Achieving all of this with such a small number of participants requires judgment, and the mix will be different for each Think Tank.

4. Think Tank Summary - Each Think Tank must produce a summary of discussion and recommendations for internal use. The summary should be done in a timely fashion, and should be done by, or have substantial input from, the outside Chair(s). Depending on the goals of the Think Tank, it may be useful to produce a meeting report or review article for publication in an appropriate journal. This should be decided in advance, in consultation with the outside Chair(s).

5. Schedule/Location - Barring unusual circumstances, Think Tanks should be held locally. The length is expected to be in the range of 1.5-2 days. The schedule of an evening session, followed by a full day, followed by a morning has become common, but should be modified as appropriate. Breakout sessions are fine, as needed.

Back to Top Tumor Immunology Think Tank

Executive Summary of the Tumor Immunology Think Tank

Three major themes emerged from the presentations and discussions at the Workshop:

1) Unequivocal evidence has emerged from a number of sources of the capacity of the immune system, alone and in combination with other modalities, to effect clinically meaningful antitumor immune responses. Specific examples include: a) the growing success of monoclonal antibody therapy (i.e., rituxan and herceptin), b) the understanding that cure of leukemias and some lymphomas by allogeneic BMT derives in large part from the antitumor response of donor T cells transferred to the patient (the so-called graft-vs.-tumor effect). In fact, GvT from donor lymphocytes is the only way to cure CML, c) dramatic antitumor effects after adoptive transfer of melanoma-specific T cells expanded ex vivo, d) antitumor effects of IL-2 in melanoma and renal cell carcinoma.

2) Recent advances in basic cellular and molecular immunology have been truly revolutionary, and have given us an unprecedented framework for understanding how the immune response is initiated and regulated, from specific cell types (i.e., dendritic cells and T regulatory cells) to specific molecules and signaling pathways. An understanding of how these pathways function and intersect, as well as how the immune system naturally interacts with developing cancers, will provide unprecedented insights and tools to effectively manipulate antitumor immunity. Already, these insights are leading to the conclusion that the most effective immunotherapies will employ combinatorial approaches that impact the antitumor immune response at multiple points.

3) Infrastructure limitation with respect to preclinical models of cancer, production of immune cells for adoptive therapy in patients, vaccine generation and availability of clinical grade recombinant molecules (i.e., cytokines, antibodies, etc.) for early phase clinical testing are severely limiting progress in the translation of the most promising immunotherapeutic combination strategies. Additionally, the growing regulatory burden for biologic therapies threatens to destroy even the current ongoing progress toward clinical translation.

Facilitation of the development and translation of rationally designed combination immunotherapy strategies should be the major NCI mandate in this area. This will require the dual approaches of empowering academically based groups for independent early stage translation as well as proactive promotion of effective public-private partnerships in this area.

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Introduction

1) Enhanced understanding of immune regulation.

It is becoming clear that the immune response is finely tuned via a set of activation and inhibitory signals expressed by critical cellular subsets. In addition to the continuously expanding knowledge base of T cell and B cell biology, a new explosion of knowledge over the past decade has occurred related to dendritic cells, NK cells, NKT cells and T regulatory cells. Each of these cell types has been shown to be central to regulation of both innate and adaptive immunity.

The myriad of cellular interactions that ultimately regulate immune responses are mediated by specific ligand-receptor interactions that in turn trigger intracellular signaling pathways. In addition to the antigen receptors on T and B cells (TCR and BCR), over 100 cytokines and cell surface molecules regulate the amplitude and quality of the output response. These ligands and receptors, many of which have been molecularly identified, appear to be roughly evenly divided between activating (e.g., IFN-α,β,γ, B7-1/2, CD28, CD40) and inhibitory (e.g., IL-10, TGF-β, CTLA-4, PD-1). Likewise, intracellular signaling pathways triggered by receptors are becoming defined in terms of how they activate or inhibit important immune effector functions as well as cellular lifespan. Indeed, more than any other system in the body, apoptosis control is a major mechanism of regulation in the immune system.

a) While much is known about individual molecules and pathways in isolation, there is much more to be learned about how these pathways interact in a coordinated fashion. Understanding the physiology of these interactions will require integration of classical molecular biology and biochemistry approaches with newer approaches in 4 areas: genomics, proteomics, systems biology and in vivo imaging.

b) Enhancement of specific activation pathways or blockade of specific inhibitory pathways has been shown to induce or exacerbate autoimmunity. Conversely, early studies using antibodies and recombinant fusion molecules has demonstrated that combinations of activating signals (such as vaccines that enhance dendritic cell function) and blockade of inhibitory signals (such as anti-CTLA-4) can dramatically enhance antitumor immunity. The development of combination approaches that simultaneously activate tumor-specific or tumor-selective immunity and block immunologic checkpoints is the most important translational mission in the cancer immunology field. Maximizing the window between antitumor efficacy and intolerable autoimmunity will require a significant investment in understanding the mechanisms of immune regulatory pathways.

2) Understanding the interaction between the immune system and the tumor microenvironment.

It is now absolutely clear that tumors express tumor-specific (from mutations and rearrangements), tumor-selective (gene expression changes due to epigenetics), and tissue-specific antigens (relevant targets for tumors derived from dispensable tissues) that the immune system can potentially recognize. If the tumor were simply an inert bag of antigens, the immune system would have no trouble eliminating all cancers. However, tumors interact actively with their environment, including the immune system. It is now emerging that an integral element of tumor biology is the immunologic effects of oncogenic changes. Examples include the inhibition of dendritic cell maturation by tumor derived factors such as VEGF and the finding that activation or inactivation of various Stat signaling pathways not only affect tumorigenesis but also have profound effects on how the immune system senses invading cancer cells. These interactions dramatically affect the balance between immune surveillance and tolerance induction. At the effector stage, it is clear that features of the tumor microenvironment, such as stromal structure and hypoxia, dramatically affect the traffic and function of immune effector cells at the metastatic site, even when appropriately activated. The mechanisms of immune interactions with the tumor microenvironment is a critical and understudied area of cancer immunology that will impact significantly on the success of immunotherapy strategies. This is a specific area that the NCI should encourage.

3) Infrastructure and regulatory barriers relevant to translation of promising immunotherapy combinations.

The diversity of immune regulatory pathways amenable to manipulation with vaccines, antibodies, and small molecule reagents offers both unprecedented opportunities and challenges for effective translation. Cell-based therapeutic opportunities, including adoptive T cell approaches, dendritic cell vaccines and bone marrow transplant-related immunotherapies, likewise offer tremendous opportunities and challenges. It is a general consensus that barriers to effective translation are mounting rather than coming down. The realization of successful cancer immunotherapy will live or die depending on whether translation is facilitated or blocked. There were 4 areas that were identified as critical to address:

a) Paucity of good preclinical mouse cancer models useful in immunological studies. Cancer immunology was largely ignored in the animal models consortium efforts despite the fact that some of the most important innovations in mouse genetics were pioneered to study the immune system in vivo. A specific effort to make the opportunities and resources of the animal model consortium directly available to the cancer immunology field is important. This will require a proactive effort on the part of the NCI.

b) Measurements of human immune responses. Although anti-tumor responses are the final arbiters in the evaluation of tumor immunotherapies, development of these therapies will only proceed in an efficient and rational manner if better means of measuring human immune responses are developed. Current methods are largely ex vivo and do not necessarily inform us about the behavior of the cells or agents in the patient. Substantial opportunities exist for NCI to actively promote the development of novel methods for the detection and measurement of the activity of the human immune system. Specific attention should be given to non-invasive imaging methods, including but not limited to PET and MRI based methods. These approaches can be used not only with labeled cells to evaluate homing to tumor sites, but also potentially for high-resolution determination of in situ lymphocyte function such as cytokine secretion or cytotoxic granule release.

c) Lack of availability of clinical grade biologic reagents to the immunotherapy community. As described above, there is a wealth of exciting biologic reagents that, if applied in proper combinations, can dramatically enhance immunotherapy potency. These range from antibodies (i.e., anti-CTLA-4), soluble ligands (i.e., soluble CD40L), and cytokines (i.e., IL7, flt-3L) to more complex recombinant viral and bacterial vaccines and finally engineered cells. Most of these are virtually unavailable to the immunotherapy community.

The three current sources for production of these reagents are invaluable, but at present not adequate to meet the increasing need.

i) RAID - while BRB/RAID is a critical mechanism to produce biologic reagents for investigators and has an extremely dedicated and expert development staff, its ability to supply these reagents is far too slow. Only a tiny fraction of RAID-approved reagents have been delivered and most that have been delivered take >3 yrs to produce. These delays are due to understaffing (staff is <10% required to complete the project portfolio relative to industry standards), tremendous bureaucratic inefficiency, lack of appropriate expertise among the review groups that select projects into the pipeline (gumming the system with flawed projects), and ineffective outsourcing and backsourcing.

ii) Institutional Processing Facilities - These facilities are becoming an important resource for institutions with highly active translational missions. However, they are extremely expensive to maintain. None of the NCI funding sources come close to adequately supporting these facilities.

iii) Companies - Biotechnology and pharmaceutical companies own a tremendous number of valuable molecules and more complex reagents at the level of patents, production expertise and actual clinical grade stocks. Most of these reagents are not available to the immunotherapy community to use in novel and promising combinations and many are not being developed at all. Much of this problem comes from the corporate culture of favoring complete control over the reagent over release of the reagent to groups that wish to utilize it in a fashion other than what the company is interested in or in combination with agents not owned by the company.

d) Regulatory barriers. FDA barriers continue to mount, driving the cost and administrative burden of doing the most innovative trials to virtually unbearable levels. The burdens are typically borne by the translational clinical investigator, who has minimal resources to meet the requirements. Some of the problem is that communication between FDA and investigators is inadequate. Much of the problem is that the NCI and the immunotherapy leadership are not appropriately educating the FDA on which safety regulations are necessary vs. frivolous, relative to the severity of the disease being treated.

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Specific Recommendations for the NCI

1) Continue to promote basic research on mechanisms of immune regulation with special emphasis on interactions between immunology and tumor biology/microenvironment. This could involve an RFA to bring together tumor biologists and immunologists to specifically address these questions. In addition, promote the development of animal models of cancer useful for the testing of immunotherapies.

2) Promote the development and application of new imaging technologies to study immune function in vivo in both animals and patients.

3) Create a mechanism for supporting collaborative, interdisciplinary consortia that is not organ site focused but rather modality focused - i.e. immunotherapy. This would greatly facilitate interactions among immunologists, cancer biologists, and clinical investigators interested in translating the most innovative and promising combination immunotherapy approaches. Inter-institutional collaborations should be emphasized with this mechanism. To provide optimal flexibility as well as emphasis on translational work, this mechanism could be based in part on the SPORE model.

4) Develop a strategic plan to effectively identify, acquire, and make available to the community the most promising immunomodulatory reagents such that their creative clinical development is most efficiently facilitated. This will involve a paradigm for interaction with the corporate world as part of the "public-private partnership."

5) Improve the availability of new biologics for immunotherapy by:

6) Develop a strategic plan to proactively interface with the FDA so that regulations are applied intelligently and flexibly and communicated in an effective and consistent fashion to clinical investigators. This should involve the recommendation of a separate review process for academically based pilot trials of combination immunotherapy approaches for patients with advanced cancer or those prognostically defined as a high probability of relapse. Also, because of the unique regulatory issues associated with biologic reagents as opposed to small molecules, consideration should be given to creating an NCI advisory/liaison group to the FDA biologics branch.

Back to Top Tumor Microenvironment Think Tank

Executive Summary of the Tumor Microenvironment Think Tank

The microenvironment in which a tumor originates plays a critical role in tumor initiation and progression, and may be an important factor in developing therapeutic approaches. The tumor microenvironment, or stroma, influences the growth of the tumor and its ability to progress and metastasize. It also can limit the access of therapeutics to the tumor, alter drug metabolism and contribute to the development of drug resistance. Because of their role in all the stages of tumor development, stromal elements represent attractive therapeutic targets. Manipulating host-tumor interactions may be important in preventing or reverting malignant conversion, and re-establishing normal control mechanisms.

Despite the importance of tumor-stromal interactions, there is a limited understanding of the stromal composition, and of the complex relationship between the tumor cells and the surrounding host cells. It is now acknowledged that tumor cells and their stroma co-evolve during tumorigenesis and progression. Stroma consists of cells, extracellular matrix and extracellular molecules. Among the identified cells are fibroblasts, glial cells, epithelial cells, adipocytes, inflammatory cells, immunocytes, and vascular cells. However, the precise nature of the cells that comprise normal stroma, how these cells or newly recruited cells are altered during tumor progression, and how they reciprocally influence tumor initiation and progression are poorly understood.

As these salient and outstanding questions are addressed, it will be possible to begin to develop complementary therapeutic strategies targeted at both the microenvironment and the tumor. Among the approaches envisioned to target the tumor microenvironment are the development of drugs that induce apoptosis or inhibit the function of the stromal cells, or the factors secreted by stroma that are required for tumor progression and metastasis. It is expected that understanding the tumor microenvironment will lead to the development of better diagnostic tests and/or improved therapeutic strategies. Finally, it may be possible to develop strategies to prevent the development of tumors based on our understanding of alterations in the microenvironment that enable tumor development.

The research priorities listed by the think tank include: a better understanding of tumor microenvironment, and the identification and characterization of the signatures of seemingly normal cells within the tumor microenvironment and signatures that reflect changes that occur as cancer cells interact with the host microenvironment. Achieving these goals can be expedited by encouraging interdisciplinary research teams and multi-institutional collaborations. Similarly, advances in technologies will be critical for progress. Among the technologies that have been identified as critical are: 1) novel in vitro 3D matrix reconstitution and organotypic models, and animal models; 2) techniques, such as laser capture microscopy, for the isolation and characterization of stromal cells; 3) the discovery of novel stromal markers through molecular profiling and their application for the development of reagents for in vivo imaging to visualize tumor-host interactions. These technologies will provide the tools for a better understanding of the tumor microenvironment and for the development of tissue- or cell-specific targeting agents. Successful approaches to respond these needs can have a dramatic effect on making the tumor microenvironment "hostile to the tumor," thereby transforming cancer into a "chronic, but benign" disease.

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Introduction

The intrinsic and extrinsic influences that transform a normal epithelial cell into a malignant cell are very complex. Enormous advances have been made over the last several decades in identifying the molecular and genetic changes of cancer cells and the pathogenesis of neoplasia. This has led to the identification of oncogenes and tumor suppressor genes and associated signaling mechanisms by which they modulate growth, survival and proliferation. These studies have generated novel therapeutic reagents such as Tamoxifen, Herceptin and Gleevec.

Stromal influence on epithelia cells begins at fertilization and continues during adulthood, wherein the microenvironment controls normal development and homeostasis. For example, macrophage association with the developing mammary gland is critical during development and CSF-1 or CSF-1R null mice (devoid of macrophage) have defective mammary glands.

Research on tumor-host interactions collectively suggests that (a) tumors are not autonomous masses of cells but function as organs composed of many interdependent cell types that contribute to tumor development and metastasis, and (b) the interactions between the tumors, the extracellular matrix (ECM) and stromal cells is bidirectional and dynamic; stromal cells include fibroblasts, adipocytes, glial cells, smooth muscle cells, and resident and recruited vascular and immune cells.

The tumor microenvironment and the malignant cells themselves constitute the tumor entity that clinicians confront when treating cancer patients. The cell-cell and cell-matrix interactions that influence the behavior of cancer cells are targets with as much potential for the development of effective therapies as the tumor cells themselves. The microenvironment can exert both positive and negative influences on tumor cells. Stromal cells can also impart stimulatory and growth inhibitory effects on tumor cells, e.g., the malignant potential of teratocarcinoma cells can be restrained during embryonic development resulting in cancer-free adult mice. Similarly, attenuation of β1 integrin (laminin receptor), EGFR or MAPK activation in highly aggressive human breast cancer cells results in a reversion of the aggressive phenotype.

The cancer cell is absolutely dependent on the stroma for its proliferation, progression, and metastasis; examples include the role of inflammatory cells (via cytokine and protease secretion) in tumor cell proliferation, angiogenesis, invasion and metastasis; the interaction of host immune cells with the vasculature; the interaction of tumor cells with the angiogenic endothelial cells, and the role of lymphangiogenesis during metastasis to the regional lymph nodes. Stromal cells can also influence organ-specific metastasis as evidenced by the role of stromal-derived cytokines and growth factors (e.g., PTHrP, CXCR4, SDF1, TGF B and RANKL) in breast and prostate cancer and multiple myeloma metastasis to bone. Finally, interaction of bone marrow stromal cells with multiple myeloma cells has been shown to contribute to the development of drug resistance.

Stroma can be targeted for therapy. Recent successes (a) in patients with multiple myeloma, where bone marrow stroma was targeted using proteasome inhibitor to attenuate bone metastasis, and (b) the development of anti-angiogenic drugs (Avastin and Thalidomide) which target the endothelial cells illustrate progress towards this goal.

The two major goals are: to obtain information about the microenvironment that would facilitate the diagnosis, prevention or treatment of cancer, and translating this information into useful clinical applications. These broad goals can be achieved through the following specific objectives: (1) identify the key components of the tumor microenvironment and define how these are altered during tumor development. Identify the stromal compartment of normal tissues and compare how these are altered in carcinogenesis. (2) Determine which alterations in the tumor microenvironment that are critical for the development, progression and metastasis; elucidate the mechanism responsible for induction of these changes. (3) Identify tumor cell stem cells and characterize the interactions between stromal cells and tumor stem cells, as well as the role of tumor stem cells in metastasis. (4) Develop therapeutic strategies to target the tumor microenvironment and interfere with site-specific metastasis by the (a) development of drugs that induce death or inhibit the function of the stromal cells that are required for progression; (b) development of reagents to target specific factors produced by stromal cells that are responsible for progression, and (c) blocking the induction of stromal factors responsible for tumor cell survival and proliferation. (5) Develop diagnostic tests to predict outcome and/or design treatment. (6) Develop strategies to prevent the development of tumors based on an understanding of the alterations in the microenvironment essential for tumor development.

These issues can be best addressed with the availability of novel technologies and model systems. Thus the development of novel in vitro 3-dimensional matrix reconstitution and organotypic models or animal models, isolation of stromal cells from normal and tumor cells using techniques such as Laser Capture Microdissection will aid in the identification of stromal markers through molecular profiling technologies. The availability of stromal markers will facilitate better reagent development for in vivo imaging to visualize tumor-host interactions as tumor cells invade and metastasize. The availability of stromal markers will also expedite the generation of reagents for tissue- or cell-specific targeting.

Significance

Critical stromal elements of the tumor are attractive targets for prevention, because they have maximal influence over tumor cells in the early stages of tumor development. As targets for therapy, they are less likely to be genetically unstable than tumor cells and thus less likely to develop drug resistance. Manipulating host-tumor interactions has the potential of reverting the malignant phenotype and establishing normal control mechanisms. Eventually, the desired goal is to reduce or eliminate metastasis-associated morbidity and transform cancer metastasis in to a chronic but benign disease.

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Specific Recommendations for the NCI

1. Establish an Interdisciplinary Tumor Microenvironment Network that includes pathologists, cancer biologists, cell biologists, oncologists, engineers, physicists, bioinformatics experts and industry representatives. Such a group will facilitate the study of normal and malignant tissue microenvironments. This can be accomplished by both centralized resources and widespread, varied funding opportunities.

2. Encourage study of the normal tissue microenvironment as a prerequisite for understanding the microenvironments of wounded tissues and tumor tissues

Establish and support a repository of normal stromal cells and matrix molecules to facilitate this research

Develop additional and improved 3-dimensional reconstitution and organotypic models that will permit the in vitro study of microenvironmental elements

Key issues in understanding normal stroma are

• What % of tumor is stroma?


• What are the various cells in the stroma?


• What are the matrix molecules?


• How does it change over time and with tumor progression?


• Genomic and proteomic analysis of tumor stroma and involvement of bioinformaticists

3. Develop a better understanding of tumor dependence on stroma

• Since experimental manipulation of stromal components in vivo has profound effects on tumor growth, progression to metastasis and immune response, there is a need for selective agents that target specific cellular and molecular stromal elements, and inhibit tumor progression.

4. Delineate the role of the microenvironment in tumor progression – key issues

• Do tumor promoters affect the stroma and facilitate metastasis?


• Are there differences between the microenvironment of a primary tumor as compared to secondary sites?


• Does the stromal compartment contribute to the "metastatic signature" of tumors?


• Are some 'stromal' cells really tumor cells in disguise?


• Can the microenvironment of tumors be effectively neutralized to inhibit progression?


• Is organ metastasis achieved solely via hematogenous spread, or do transiting cells initially exit via lymphatics and subsequently spread hematogenously?


• What is the role of inflammatory cells during invasion, migration, tumor growth and metastasis? To achieve this it will be necessary to study inflammation in vivo, in relevant immune-competent (mouse) models of progression (transgenic or syngeneic xenograft).


• Do stromal elements interact or modulate the tumor stem cell 'niche' and do these interactions change as tumor progress and metastasize?

5. Stromal Genetics – key issues

• What is the role of host genetics in influencing the stromal formation?
• Does stromal composition differ in different inbred mouse strains?
• Do epigenetic changes influence stromal composition?

6. Encourage the use of existing technologies for visualizing the components of the stroma at the level of individual cells and molecules

• Expand access to multiphoton microscopy and deconvolution microscopy to study the microenvironment.


• Develop novel imaging technologies to study tumor microenvironment; recruit/collaborate with engineers and physicists who can apply the most current imaging technologies to the study of the normal and tumor microenvironment.

7. Translation of basic knowledge to human disease

• Discover and validate therapeutic targets derived from the tumor microenvironment


• Encourage mouse and human research to improve our chances of discovering useful targets.

8. Role of NCI

• Establish a network of interconnected, multidisciplinary investigators and collaborative groups to work together on understanding the tumor microenvironment, facilitated by both centralized resources and widespread, varied funding opportunities.

Such an infrastructure should

• Provide centralized administrative support


• Provide and maintain repositories


• Produce and distribute reagents


• Provide core facilities


• Facilitate interdisciplinary collaborations


• Train scientists

• Animal models / fluorescent markers


• Imaging (multiphoton) at one micron resolution


• Spectral deconvolution microscopy


• Selection of live cells from tumors


• 3D matrix reconstitution


• Organotypic models

• In individual labs


• In "Centers of Excellence"


• In central or decentralized cores


• At meetings and conferences


• At courses on the tumor microenvironment

9. Establish and encourage a Systems approach to the study of the tumor microenvironment

10. Encourage interactions with other NCI-supported multidisciplinary groups such as EDRN, MMHCC, SPOREs, and Cooperative Groups

11. Encourage interactions with other ICs with similar interests (NIEHS, NIDDK, NHLBI, NIAMD, and NIDCR)

Back to Top Tumor Stem Cell & Self-Renewal Genes Think Tank

Executive Summary of the Tumor Stem Cell & Self-Renewal Genes Think Tank

The Tumor Stem Cell and Self-Renewal Genes Think Tank was convened to identify the major scientific issues in the field and provide recommendations to the NCI to help advance research in this area. The participants came to the following scientific conclusions:

• Efforts are required prospectively to isolate and purify the tumor initiating cells (T-IC) from a larger variety of hematological and solid tumors. Since many tissues do not have as rich a source of cell surface markers as the blood system, effort will need to be expended to develop the means for T-IC purification.


• The normal tissue stem cell is a likely target for the initial carcinogenic insult, because it has the long life necessary for accumulating the multiple genetic or epigenetic changes required for malignant transformation.


• Although the events required for malignant transformation may all accumulate in the normal tissue stem cell, this cell does not necessarily become the tumor stem cell.


• It appears that the genetic and biochemical pathways regulating the "stem" phenotype in normal stem cells are subverted later to maintain the tumor stem cell phenotype.


• There is evidence to suggest that epigenetic mechanisms can produce an inheritable tumor stem cell phenotype. Epigenetic control also underlies a great amount of stem cell regulation, as exemplified by the polycomb gene Bmi-1. Thus, there is a link between normal stem cell regulation and the control of cancer stem cells.

Finally, it was recommended that the National Cancer Institute:

• Continue support for opportunistic basic research into tumor stem cell biology.


• Develop a Research Consortium to encourage transdisciplinary approaches and to provide specialized research reagents.

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Introduction

The clonal nature of most malignant tumors is well established. Experiments spanning several decades have shown, however, that as many as 10⁶ murine or human tumor cells are required to transplant a new tumor from an existing one. Two theories have been developed to account for the observation that apparently not every tumor cell is a tumor initiating cell (T-IC). The stochastic theory predicts that every tumor cell can form an entirely new tumor; however, entry into the cell cycle is a stochastic event with low probability. Alternatively, tumor cells may exist in a hierarchical state in which only a small number of cells possess tumor initiating potential. If the stochastic model is correct then tumor cells are biologically homogeneous and genetic or epigenetic programs that allow for tumorigenesis are operative in the majority of cells that comprise a tumor. The hierarchical model, however, predicts the tumor cells possess a functional heterogeneity and that quantitatively the cells capable of tumorigenesis are a relatively minor population among the bulk of tumor cells.

Recent data from both hematologic malignancies and solid tumors have suggested that there are only minor populations of cells in each malignancy that are capable of tumor initiation. These tumor initiating cells have the functional properties of a tumor stem cell. They appear to be capable of asymmetric division and self renewal, and are only a minor faction among the bulk of more differentiated cells in the tumor. These observations have profound implications for tumor biology research as well as successful tumor therapy. The Tumor Stem Cell Think Tank addressed several of the outstanding scientific questions that will define research in this area, as well as the needs of the research community to promote progress in this research.

(1) What is the current evidence for tumor initiating stem cells among tumors arising from a variety of tissues?

Currently, tumor stem cells have been isolated and characterized in several hematologic malignancies and two solid tumors. The critical experimental design that underlies all these studies is the development and use of a functional assay for tumor establishment and the prospective isolation of the T-IC. Often normal tissue stem cell markers are used to identify these populations, but a functional assay such as transplantation of human leukemic stem cells into immunodeficient murine models such as the NOD/SCID mouse is most important in identifying tumor initiating cells. One of the first tumors in which a stem cell was identified was acute myeloid leukemia (AML). In this disease, the frequency of the leukemic stem cell (LSC) was approximately 1 per million AML blasts, establishing that not every AML cell had LSC capacity. A CD34⁺, CD38^- cell fraction representing 0.1-1% of the tumor cells possessed all the leukemia initiating activity in the NOD/SCID model. By contrast, the CD34⁺, CD38⁺ cells and the CD34^- cells, which comprise most of the cells in the tumor, could not initiate leukemia. A multiple myeloma stem cell has also been characterized. Multiple myeloma cell lines and primary patient derived cells express the cell surface marker syndecan -1 (CD138). Expression appears during the course of B-cell differentiation. A population of cells representing <5% of the cells in the bulk population of multiple myeloma cells were found to be CD138^- and possessed in vitro clonogenic potential. These cells also engrafted successfully into NOD/SCID mice, whereas CD138⁺ cells did not engraft. CD138^- cells were also CD19⁺ and CD20⁺, and they expressed higher levels of KI67 (a cell proliferation antigen) than CD138⁺ cells. Recently a mammary carcinoma stem cell has been isolated primarily using three cell surface markers (CD44, CD24, and epithelial specific antigen). The tumor initiating capacity of the cells was verified in a NOD/SCID engraftment assay, and the T-ICs represented only 2% of the unfractionated cells.

Finally, a putative brain tumor stem cell has also been isolated. These cells appear to be between 0.3 - 25 % of the cells in the brain tumors examined. They are positive for the neural stem cell marker CD133 and have a marked capacity for self renewal and differentiation. Transplantation of these putative neural tumor stem cells into the forebrains of NOD/SCID mice yields tumors phenotypically resembling the tumors from which the stem cells were isolated.

The think tank participants arrived at a consensus that:

A. Isolation of stem cells from more hematological malignancies and solid tumors is required to validate the general nature of the presence of stem cells in tumors.

B. Functional assays, such as NOD/SCID mouse engraftment and single cell tumor initiating capacity assays, are necessary to establish the true stem nature of isolated cells. Cell surface morphological markers alone are insufficient to accurately characterize stem cells.

C. Efforts are needed to isolate prospectively and purify the T-IC. Since many tissues do not have as rich a source of cell surface markers as the blood system, effort should be expended to develop the means for T-IC purification.

(2) Is the tumor stem cell a derivative of a normal tumor stem cell or a later more differentiated progenitor cell?

It is unclear whether tumor stem cells arise exclusively from normal tissue stem cells, or from progenitors that have differentiated from the stem cell itself. From a theoretical standpoint, it has been well established that neoplasia arises as a consequence of the acquisition of multiple oncogenic events. Thus, the initial events must occur in cells that persist. Hierarchical differentiation of blood cells from a stem and progenitor population has been well studied and characterized in the hematopoietic system. It is thus not surprising that a great deal of evidence on the nature of the tumor stem cells is available from studies of hematopoietic malignancies. In AML, the stem cell population appears to share many of the markers of the normal hematopoietic cell (e.g. CD34⁺, CD38^-, HLA-DR^-); however the leukemic stem cell appears to overexpress the IL-3 Rα subunit (CD123) relative to normal hematopoietic stem cells (HSC). The similar cell surface properties, self-renewal properties, and complexity of stem cell hierarchies, as determined by clonal stem cell tracking of both LSC and normal stem cells, suggest that they are closely related and have been interpreted to suggest that LSC are derived from HSC. In the blood system, only the stem cells have the long-life required to accumulate all the initiating mutations and these could be passed to the progeny that are continuously produced from such stem cells.

In experimental murine systems, it has been found that the more committed myeloid progenitor cells, especially those employing the MLL oncogene, are also capable of becoming tumor stem cells. This gene codes for a transcription factor that regulates Hox gene expression. The Hox genes have been implicated in stem/progenitor cell expansion and self renewal. MLL fusion proteins created by chromosomal translocations are frequently associated with acute lymphoid and myeloid leukemias. Transduction of an MLL fusion protein into purified populations of either hematopoietic stem cells or more committed granulocyte macrophage progenitors gives rise to cells that produce a rapid AML. Although the disease is similar with both populations, the stem cell fraction is much more potent. Thus, a more differentiated cell can also act as a tumor initiating stem cell if the genetic alteration endows this cell with self-renewal capacity. In chronic myelogenous leukemia (CML), the t(9;22) translocation joining the BCR and ABL genes is found in hematopoietic stem cells, but the mRNA and protein for the fusion gene are found only in later progenitors. In AML patients carrying the AML-1 Eto translocation in hematopoietic stem cells, the stem cell fraction is still capable of normal differentiation, but the more committed cells possess clonogenic leukemia potential only, suggesting that the AML/ETO translocation created a pre-leukemic stem cell. Additional alterations occurred in the more committed cells to create a frank leukemia. Thus, the tumor stem cell can arise in hematologic malignancies from multiple alterations occurring in normal stem cells, or the initial alteration could occur in the stem cell, with additional hits occurring in a more differentiated progenitor cell. Finally, it is also possible that under some rare circumstances, the initiating event could occur in the committed progenitor to convert it into a self renewing stem cell. It will be important to determine if the leukemogenic pathways obtained in experimental murine systems are recapitulated in the human disease.

Mammary tumor stem cells are CD44⁺, CD24^-, and epithelial stem antigen positive, and this phenotype overlaps with that of epithelial stem cells. Definitive evidence, however, that these cells arise from the normal mammary tumor cells is thus far lacking.

The tumor stem cell isolated from a variety of brain tumors, including slowly proliferating astrocytomas and highly malignant medulloblastomas and glioblastomas, contain tumor stem cells that express the CD133 antigen and nestin found on normal neural tumor stem cells. The neural tumor stem cell does not contain any of the markers characteristic of more differentiated neural cells. Again, these data suggest that the normal neural stem cell is the precursor of the brain tumor stem cell. This conclusion about neural tumors is supported by molecular genetic studies performed in neural cells. Simultaneous knockout of p53 and NF-1 in neuronal precursor cells results in the development of brain tumors. Imaging studies demonstrate that the tumors arise in two areas: the subventricular zone of the lateral ventricle and the hippocampus. These areas of the brain have been previously shown to be reservoirs of normal neural stem cells. Studies of the generation of neurofibromas from Schwann cells have also shown that primitive neural crest cells can become tumor stem cells. The transcription factor Krox20 participates in the differentiation of these Schwann cells, but has recently also been shown to become expressed in the primitive neural crest. Homozygous deletion of the Krox20 gene using a Krox20-Cre system causes some of the deleted cells to become neurofibromas. It is postulated that the cells must accumulate other stochastic events (e.g., loss of NF-1) to become fibromas, because all the cells do not become transformed.

The participants concluded the following:

A. Among the solid tumor stem cells identified there is an overlap between the phenotype of the normal tissue stem cell and the tumor stem cell, but definitive evidence as to whether tumor stem cells are actually transformed normal stem cells is not yet available.

B. In the hematologic and neural malignancies there is evidence that tumor stem cells may be heterogeneous. Some may arise from the normal stem cells population, but others may arise from more differentiated progeny cells that have acquired self renewal capacity.

C. The normal tissue stem cell is a likely target for the initial carcinogenic insult, because it has the long life required to accumulate the multiple genetic or epigenetic changes required for malignant transformation.

D. Although the events required for malignant transformation may all accumulate in the normal tissue stem cell, this cell does not necessarily become the tumor stem cell.

(3) What genetic pathways may be important in maintaining the tumor stem cell state?

The proteins involved in self renewal in normal tissue stem cells appear to be subverted in tumorigenesis to allow the tumor initiating cells to maintain self renewal capacity. Two families of proteins related to self renewal were considered in detail: the poycomb gene Bmi-1 and the Wnt signaling pathway proteins. The polycomb genes have an essential role in embryogenesis, regulation of the cell cycle and lymphopoiesis. These genes are essential for the silencing of other families of genes. It has been shown by RT-PCR analysis that knockout of the polycomb gene Bmi-1 in mice results in a progressive loss of all hematopoietic lineages. This loss results from the inability of the Bmi-1 ^(-/-) stem cells to self renew. Bmi-1 ^(-/-) cells displayed altered expression of the cell cycle inhibitor genes p16^INK4a and p19^ARF, and down regulation of a gene coding for an inhibitor of apoptosis. The p16 and p19 proteins interact with the p53/Rb regulated cell cycle pathways. Introducing genes known to produce acute myeloid leukemia (AML) into Bmi-1^(-/-) hematopoietic stem cells (fetal liver cells) induced AML with normal kinetics. However, the Bmi-1^(-/-) leukemic stem cells from primary recipients were unable to produce AML in secondary recipients. These results demonstrate that Bmi-1 is also required for self renewal of leukemic stem cells in AML.

Another group of genes involved in self renewal are those involved in the Wnt signal transduction cascade. The Wnt protein binds to a receptor called Frizzled and activates cell fate decisions during tissue development. It has been shown that deletion of the TCF-4 gene, a transcription factors at the end of the Wnt signal transduction cascade, causes early neonatal death in mice. The mice lacking the gene have a single histological defect - the intestinal stem cell lining is absent. It has also been shown that inhibitors of Wnt signaling leads to inhibition of hematopoietic stem cell growth in vitro and reduced hematopoietic reconstitution in vivo. Activation of Wnt signaling in hematopoietic stem cells leads to increased expression of Hox B4 and Notch-1 genes previously replicated in self renewal of hematopoietic stem cells. The Wnt signaling pathways has been shown to be involved in both hematopoietic malignancy and colon carcinoma.

Although the Wnt ligands themselves are only rarely involved in tumorigenesis, mutations mimicking Wnt receptor (Frizzled) activation induce a set of genes associated with repression of differentiation and potentiation of self renewal. In general, these mutations involve Wnt signal transduction proteins: activation of β-catenin and inactivation of the (APC) adenomatosis polyposis coli protein. In myeloid leukemia, non-phosphorylated β-catenin accumulates in granulocyte macrophage progenitors as they progress toward leukemia. These normally more committed progenitors can thus acquire self renewal properties. A similar accumulation of non-phosphorylated β-catenin has also been observed in multiple myeloma cells. In colon cancer, the APC gene is mutated early in the development of 90% of colon carcinomas. Similarity in gene expression patterns between populations of colon cancer cells and colon epithelial stem cells has also been observed by DNA microarray analysis. It is possible that mutations in the Wnt signaling pathway maintain the program of stem cell genes in the "on" position.

Two other proteins that may play a role in tumor stem cell biology are nucleostemin and the tumor suppressor PTEN. Nucleostemin is abundant in self renewing cells such as mouse embryonic and neural stem cells as well as several human cancer cells. Although the exact function of nucleostemin is not yet known, it behaves like a molecular switch to control cell division, perhaps through binding to p53. Knockout of the PTEN phosphatase in prostate cancer cells allows the expression of genes associated with metastasis. As metastatic cells are likely prostate tumor stem cells, it is likely this gene may regulate expression of stem cell related genes.

In conclusion, it appears that the genetic and biochemical pathways regulating the "stem" phenotype in normal stem cells are subverted to maintain the tumor stem cell phenotype.

(4) Can the tumor stem cell phenotype be epigenetically programmed or reprogrammed?

Human tumor cells often demonstrate abnormal patterns of DNA methylation. DNA methylation provides an epigenetic mechanism for altering gene expression by silencing genes. Hypermethylation frequently underlies the silencing of tumor suppressor genes. The opposite condition, in which DNA is hypomethylated, often in concert with regional hypermethylation, has been observed in a spectrum of human tumors. That such hypomethylation can have a fundamental effect on tumor cell development has recently become clear. Transgenic mice that are heterozygously deleted for a DNA methyltransferase show a substantial decrease in total genomic methylation in all tissues. At 4 to 8 months of age, these mice develop an aggressive T-cell lymphoma with a high frequency of trisomy in Chromosome 15. There appears to be a link between DNA hypomethylation and chromosomal stability. Chromosome 15 is frequently duplicated in T-cell lymphomas and the c-myc oncogene is located on this chromosome. c-Myc is overexpressed in many of the hypomethylated tumors and in T-cell lymphomas as well.

Further evidence that hypomethylation may have a causal role in carcinogenesis was obtained by crossing the DNA methylase heterozygote mice with mice prone to develop soft tissue sarcoma with simultaneous loss of heterozygosity (LOH) of the NF-1 and p53 genes. The resulting transgenic mice develop sarcomas at an earlier age and demonstrate an increase in LOH in the hypomethylated versus the normally methylated cells. The increase in the rate of LOH is the result of a specific effect of hypomethylation on the stability of pericentric and centromeric chromosomal regions.

Other experiments with nuclear cloning provide evidence that the tumorigenic phenotype of tumor stem cells can be reprogrammed epigenetically. Introducing the nucleus of a murine melanoma cell into an enucleated murine ovum with subsequent transfer into a surrogate female resulted in a normal offspring. The neural crest derived cells including melanocytes were all normal in the resulting mouse. Thus, the micro-environment induced a genetic reprogramming. However, the genetic mechanisms that existed in the tumor nuclei were maintained as these mice had a high incidence of neoplasia. Thus, both genetic and epigenetic mechanisms may be active in neoplastic development. This result with melanoma was a rare experimental success among a number of tumor nuclei tested, and the reasons for the lack of repeated success with nuclei from other tumors is under investigation.

That the microenvironmental niche that a cell resides in can have profound effects on the cellular phenotype is also demonstrated by studies on the fate of embryonic stem cells derived from the blastocyst inner cell mass. When they are transplanted back into another embryo, they maintain their normal phenotype. If, however, they are transplanted into a differentiated microenvironment such as kidney or liver, the cells form a teratoma.

In conclusion, there is evidence to suggest that epigenetic mechanisms can produce an inheritable tumor stem cell phenotype. Epigenetic control also underlies a great amount of stem cell regulation as exemplified by the polycomb gene Bmi-1, thus providing a link to the control of cancer stem cells.

Future Directions:

At the conclusion of the Think Tank, a number of important future basic research questions were synthesized from the scientific discussion. They are summarized below:

1) What governs the rate of proliferation of stem cells and in particular tumor stem cells? A corollary question would be whether the size or quality of the stem cell microenvironment (niche) acts as a constraint on stem cell growth?

2) What creates the stem cell niche for a tumor stem cell (i.e. do tumor stromal cells constitute the tumor stem cell niche?)

3) Does the transition from semi-linear to exponential cell growth occur when the tumor cells become independent of the stromal niche?

4) Can oncogenes and their associated mutations affect asymmetric versus symmetric divisions in stem cells?

5) Is the object of Darwinian selection in the tumor the T-IC, rather than the more differentiated tumor cells? It is likely that alleles for malignancy spread in a tissue because they arise in stem cells and provide advantages to the tumor stem cells that carry them.

6) Are the phenotypes of invasion and metastasis uniquely connected to the tumor stem phenotype?

7) Stem cell quiescence versus growth and differentiation must ultimately be understood in terms of progression through the cell cycle. It will be important to determine whether the retinoblastoma (Rb) gene product is as critical in this process as it appears. Rb plays a key role in cell cycle progression and differentiation in a number of tissues. Hypophosphorylation of Rb forces cells to leave the cell cycle and enter G₀, therefore regulation of this protein is likely to play a role in regulating true stem cell state. This is substantiated by studies on the Bmi-1 gene and the genes it regulate that interact with the Rb/p53 cell cycle pathway.

8) Most human carcinogens are strong tumor promoters and weak initiators of carcinogenesis. If tumor promoters work by increasing the size of the target population, then they must work by increasing the population of already initiated cells. Thus, it is important to understand whether tumor promoters work on initiated epithelial stem cells or on stromal cells, as the stromal cells may control the size of the stem cell niche.

9) Can the roles of mutation and epigenetic mechanisms be distinguished in the generation of the tumor stem cell phenotype?

In addition a number of questions related to the future of cancer therapy were also considered.

1) Can the current benchmark for measuring the success of cancer therapy, tumor shrinkage, be changed to something more biologically relevant? The success of therapy can only really be measured by understanding the effect of the therapy on the tumor stem cell. Unfortunately this is difficult to obtain because of the lack of tumor stem cell markers.

2) Can we develop xenograft models that recapitulate the stem cell transition to more differentiated progenitors in a tumor? Such models might be useful to begin understanding the effects of therapy on tumor stem cells.

3) Are there methods for treating tumors that might cause a collapse of the stem cell niche? It is likely that most human tumors depend on stromal cells that define the niche and can control the size of the stem cell niche.

4) Are the cells that form the tumor stem cell niche different from those forming the normal tissue stem cell niche? Might any differences present an opportunity for directing selective therapy to tumor stromal cells?

5) Finally, might tumor stem cells be more or less sensitive to apoptosis inducing stimuli? Such information is critical in designing therapies that destroy the tumor stem cells responsible for continued expansion of the tumor and subsequent metastasis.

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Specific Recommendations for the NCI

Technical Recommendations:

1) Develop methods to encourage surgeons involved in clinical research to collect tumor specimens directly into trypsin disaggregation solutions and provide viable freezing of cells in large banks to allow scientists to isolate unselected populations of tumor stem cells.

2) Encourage the development of real time PCR technology to examine gene expression in tumor stem cells. This technique appears to give more valuable information than DNA microarray technology for analysis of stem cells.

3) Improve in vivo and in vitro functional assays for tumor stem cells to allow for more accurate identification of these cells in concert with cell surface phenotype identification.

4) Improve in vivo organotypic assays to understand symmetric versus asymmetric cell division.

Administrative Recommendations:

1) Continue National Cancer Institute support for opportunistic basic research into tumor stem cell biology.

2) Develop a Research Consortium to facilitate transdisciplinary approaches and to provide specialized research reagents that will advance research in this area.

Back to Top Cell Decisions in Response to DNA Damage: Survival vs. Programmed Cell Death Think Tank

Executive Summary of the Cell Decisions in Response to DNA Damage: Survival vs. Programmed Cell Death Think Tank

Current cancer therapy relies heavily on DNA damaging agents (radiation, DNA alkylating agents, etc.) to induce programmed cell death in cancer cells. The proven success of this therapy, albeit only in some patients, implies that cancer cells are more sensitive to killing by DNA damaging agents than normal cells. This increased sensitivity is believed to be due largely to defects in DNA damage response pathways within the cancer cell. Compelling experimental evidence suggests that it is possible to dramatically modulate the sensitivity of cells to DNA damage. Similarly it may be possible to modulate the cell-specific and stimulus-specific responses to DNA damage leading to cell death. These responses vary dramatically between cell and tissue type, metabolic state and genetic background. If the sensitivity of cancer cells to DNA damaging agents and the cell death response to them could be specifically increased (or the sensitivity of normal cells to these agents be specifically decreased) by just one order of magnitude this would lead to a significant increase in cancer cure rates.

The obvious benefits of such an outcome are:

• reduced collateral tissue damage from the use of lower doses of radiation or chemical agents that would be required to kill cancer cells,


• reduced risk of second cancers from cells irradiated at the edges of the therapeutic radiation field,


• improved tumor targeting by combining the focusing power of radiation therapy with pharmacologic enhancement of tumor susceptibility to DNA damage.

A major goal of the workshop was to identify the knowledge and resources needed to optimize the DNA damage response leading to programmed cell death in human cancer. The Think Tank participants put forth a number of recommendations that can be summarized into 4 broad areas:

• Support systems biology analyses of the human DNA Damage Response (DDR) networks including the complete mapping of the biochemical and regulatory circuitries that link the cell-cycle checkpoints, apoptotic, and DNA repair pathways with the DDR.


• Support further identification of molecular targets for enhancing programmed cell death in response to DNA damage, particularly by investigating p53-independent pathways of DDR-induced cell death and by investigating strategies to modulate activity of DDR sensor and mediator proteins to amplify cell death signals.


• Develop reagents to study the DDR in vivo, particularly a resource library of phosphospecific antibodies and DDR read-out reagents for quantitative and dynamic measurements.


• Convene a conference focused on enhancing molecular pathology approaches for the analysis of DDR in human tissues and undertaking comparative studies of the DDR in normal and tumor cells/tissues to elucidate novel anti-apoptotic components altered in cancer cells.

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Introduction

The introduction of DNA damage is a major therapeutic strategy for killing cancer cells. However, cell death is not the sole option for cells in response to DNA damage. In brief, the damaged cancer cell has two options: To die by regulated programmed cell death (PCD), or to survive by preventing cell division until DNA repair can be completed. In their initial stages, both PCD and survival/DNA repair processes initiated by DNA damage share the same signaling cascades based on high-level protein kinases (e.g., ATM, ATR) and secondary kinases (e.g., Chk1 and 2) along with a number of other proteins involved in signal detection and "mediation" of signaling and repair. What is less clear, however, is how, at the molecular/mechanistic level, human cancer (and also normal) cells that have sustained DNA damage assess its severity and make the ultimate cellular decision between death and survival. The aim of this Think Tank was to advance understanding of the mechanisms involved, with the ultimate goal of finding strategies that will select or enhance cell death.

Think Tank Program
Session I: DNA Damage and Repair

Discussion topics included:

• How do human cells (normal and cancer) detect primary DNA damage?


• How are signals of primary DNA damage amplified by the cell?


• What are common and distinct responses to different types of DNA lesions?

The most lethal type of DNA damage is DNA double strand breaks (DSBs). DSBs can be caused directly by radiation or indirectly after DNA modified by chemotherapeutic drugs is processed by cellular enzymes. Both radiation and DNA-modifying chemotherapeutic drug