**[Table of Contents]{.underline}**

**Summary
Report**..........................................................................................3

**Tumor Immunology Think Tank**

Executive
Summary...............................................................................15

Introduction.........................................................................................16

Recommendations.................................................................................19

**Tumor Microenvironment Think Tank**

Executive
Summary...............................................................................21

Introduction.........................................................................................22

Recommendations..............................................................................\....24

**Tumor Stem Cells and Genetic Basis of Self-Renewal Think Tank**

Executive
Summary...............................................................................27

Introduction.........................................................................................28

Recommendations.................................................................................35

**Cell-Decision in Response to DNA Damage: Survival vs. Programmed**

**Cell Death Think Tank**

Executive
Summary...............................................................................37

Introduction.........................................................................................38

Recommendations.................................................................................45

**Cancer Etiology: Role of Exogenous and Endogenous Chemicals Think
Tank**

Executive
Summary...............................................................................47

Introduction.........................................................................................48

Recommendations.................................................................................55

**Epigenetics Think Tank**

Executive
Summary...............................................................................57

Introduction.........................................................................................58

Recommendations............................................................................\......71

**Inflammation and Cancer Think Tank**

Executive
Summary...............................................................................73

Introduction.........................................................................................74

Recommendations..............................................................................\....78

**Cancer Susceptibility and Resistance Think Tank**

Executive
Summary...............................................................................79

Introduction.........................................................................................80

Recommendations...............................................................................\...91

**Rosters**

Think Tank Participant
Rosters..................................................................93

Division of Cancer Biology Staff and
Organizers...........................................119

Acknowledgements....................................
.........................................121

**Think Tanks in Cancer Biology**

**Summary Report**

**I. [What we sought to accomplish]{.underline}**

A year ago, Dr. von Eschenbach charged 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\) **The 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 Cells and 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.
Apoptosis** - 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.

**II. [How the Think Tanks were organized and run]{.underline}**

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.

**III. [Overall recommendations]{.underline}**

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]{.underline}*

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]{.underline}
[vivo]{.underline} 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.

The tumor microenvironment has become an active area of investigation in
the NCI R01 grant portfolio, a recent development underscored by the
creation of a new CSR Study Section called Tumor Microenvironment. The
Think Tank participants emphasized the importance of continuing this
support, but also recommended the formation of a network or alliance to
encourage cooperative, interdisciplinary studies beyond the scope of R01
grants. The Tumor Microenvironment Network would bring investigators
experienced in this area together with scientists with complementary
expertises. It would leverage existing grant support, but provide
incremental funding for cooperative projects and for the creation of
freely accessible, common resources that would benefit the entire
research community. The goals of the Network would be to:

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]{.underline}*

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]{.underline}*

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]{.underline}*

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]{.underline}*

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.

**[IV. Concluding Remarks]{.underline}**

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.

**[APPENDIX A]{.underline}**

**Guidelines for Organization of Think Tanks**

1.  [Think Tank Goals]{.underline}- 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]{.underline}- 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]{.underline}- 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]{.underline}- 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]{.underline}- 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.

**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]{.underline} [vivo]{.underline}, 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]{.underline}.

**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.

**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, Β7−1/2, 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]{.underline}.

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]{.underline}. 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]{.underline}.

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]{.underline}. 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]{.underline} [vivo]{.underline} 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]{.underline}
    [situ]{.underline} 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]{.underline} -- 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]{.underline} -- 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]{.underline} -- 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.

> **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]{.underline} [vivo]{.underline} 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:

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a)  Convening a blue ribbon panel to review BRB/RAID that will be
    charged with developing specific recommendations on how to enhance
    the efficiency, quality and speed of reagent production.

b)  Developing a mechanism to support infrastructure for the most active
    institutionally based facilities committed to cellular and biologic
    reagent production for biologic therapy of cancer.

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```
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.

**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.

**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.

**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]{.underline}*, 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?

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-   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

What kind of technologies should NCI support?

-   Animal models / fluorescent markers

-   Imaging (multiphoton) at one micron resolution

-   Spectral deconvolution microscopy.

-   Selection of live cells from tumors

-   3D matrix reconstitution

-   Organotypic models

Where would such facilities reside?

-   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)

**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.

**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?]{.underline}

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?]{.underline}

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 acquistion 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 hemapoietic
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
hematopoeitic stem cells, but the mRNA and protein for the fusion gene
are found only in later progenitrors. 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 commited 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 Krox 20 gene using a Krox 20 -- 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?]{.underline}

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 lymphopoieisis. 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 p19ARF, 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(-/-)
hematopoeitic 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]{.underline}
[vitro]{.underline} and reduced hematopoietic reconstitution
[in]{.underline} [vivo]{.underline} . Activation of Wnt signaling in
hematopoeitic 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 ß- catenin and inactivation
of the (APC) adenomatosis polyposis coli protein. In myeloid leukemia,
non-phosphorylated ß-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 ß-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?]{.underline}

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 microenviroment 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:]{.underline}

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 Go, 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.

**Specific Recommendations for the NCI:**

[Technical Recommendations:]{.underline}

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]{.underline} and [in vitro]{.underline} 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]{.underline} organotypic assays to understand
symmetric versus asymmetric cell division.

**[Administrative Recommendations:]{.underline}**

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.

**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.

**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 Amediation@ 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 drugs are currently
used to treat human cancer.

The response to a DNA DSB includes DNA repair and DNA damage signaling
components. DNA repair pathways are clearly important; however, DNA
repair genes are not frequently mutated in human cancer and it is not
clear that inhibiting these pathways will lead to cancer-specific
killing. DNA damage signaling pathways (also known as DNA damage
checkpoint pathways) include sensors, which detect the presence of DNA
DSBs, transducers, which produce a DNA damage signal, and effectors,
which induce cell death or cell cycle arrest (transient or permanent).
Mutations that inactivate DNA damage checkpoint genes are extremely
prevalent in human cancer, resulting in rewiring of the DNA damage
signaling pathways and differences in the response of cells to DNA
damage.

In the last ten years there has been tremendous progress in our
understanding of DNA damage checkpoint pathways. Most of the known DNA
damage checkpoint genes were cloned within the last ten years. Their
function is just beginning to be understood and key research
discoveries, especially with regard to the DNA DSB sensors, were
published in the last year or are still in the process of being
published. The emerging theme is that there is a small number of DNA DSB
sensors and the response of the cell to DNA damage (cell cycle arrest or
cell death) depends on which sensor is being used to recognize the
damage. Crystallography analysis suggests that recruitment of at least
one sensor (53BP1) to DNA DSBs is amenable to pharmacologic
intervention. Inhibition of 53BP1 recruitment to sites of DNA DSBs would
result in increased recruitment of other sensors (specifically, the
NFBD1/MDC1-Mre11 complex), which is thought to be more potent in
promoting cell death in response to DNA DSBs.

Cells also have at least two transducers of the DNA damage signal in the
form of the protein kinases ATM and ATR. How these kinases are activated
by the DNA DSB sensors is not yet clear. The pathway downstream of ATM
and ATR is somewhat better understood, although there are still gaps in
our knowledge there as well.

In conclusion, how cells detect the presence of DNA DSBs and transduce
the DNA damage signal represents a major gap in our knowledge. The
recent progress in this field suggests that we are at the verge of
making considerable progress. Characterization of the DNA DSB sensors
and transducers is expected to identify promising and pharmacologically
amenable targets for development of cancer therapeutics.

**Session II: Checkpoints and Apoptosis**

Discussion topics included:

-   How do cells assess DNA damage information to decide between
    survival and death?

-   Is DNA repair linked to either checkpoint or apoptosis induction?

-   Are DNA lesions sufficient to trigger apoptosis?

The p53-dependent pathway which induces apoptotic cell death in response
to DNA damage is clearly relevant in terms of cancer therapy toxicity,
since normal tissues exposed to DNA damage can undergo apoptosis.
Stabilization of p53 following DNA damage results in its accumulation in
both nuclear and cytosolic compartments. Cytosolic p53 can directly
activate the proapoptotic activity of Bax to permeabilize the
mitochondrial outer membrane. It remains to be determined to what extent
this cytosolic effect, independent of transcription, contributes to
p53-mediated death. If cytosolic p53 is apoptotic, the transcriptional
upregulation of PUMA may provide another block to the anti-apoptotic
functions of Bcl-2/Bcl-xl. However, in most solid tumors these pathways
are unlikely to contribute to DNA damage-induced death, because in most
tumors p53 is inactivated by mutations. Therefore it is anticipated that
inhibiting p53-dependent apoptosis may enhance the therapeutic index of
cancer therapy, although there are still reservations regarding whether
such an approach would be beneficial in humans.

p53-independent cell death pathways are likely to be of greater
relevance to killing of most solid tumors in response to therapy. Cell
death in p53-mutant cancers may be due to apoptosis or perhaps more
likely to progress through the cell cycle with unrepaired DNA DSBs,
which occurs when DNA damage checkpoint genes are mutated. While in
yeast and humans it is well established that defects in DNA damage
checkpoint genes lead to extreme radiosensitivity (for example, in
ataxia-telangiectasia patients in which the ATM transducer is mutant), a
link between mutations in DNA damage checkpoint genes in human cancers
and their radiosensitivity has not been systematically investigated
(with the exception of the effect of p53 mutations). Recent studies have
identified a p53-independent apoptotic pathway, involving p53-related
p63 and p73, in mediating DNA damage induced apoptosis. Activation of
p73 by genotoxic agents has been shown to involve the nuclear c-Abl
tyrosine kinase and contributes to the apoptotic response to
p53-negative cells. But in general, it is fair to say that we do not
understand well how DNA DSBs kill cancer cells that bear mutant p53.

In addition, apoptosis is not the only way for damaged cells to die.
Excessive or persistent damage can trigger other modes of cell death,
including necrosis and mitotic catastrophe. It is also possible that
these modes of cell death could offer opportunities for new therapeutic
strategies.

**Session III: DNA Damage and Cancer Therapy**

Discussion topics included:

-   How can our knowledge about DNA damage responses be best exploited
    to therapeutic benefit?

-   What are the current roadblocks to translating our knowledge about
    DNA-damage responses to therapeutic strategies?

Tumor cells start out as intrinsically more sensitive to apoptosis by
virtue of their obligate oncogenic lesions, which push cells closer to
their apoptotic activation threshold. This is thought to explain the
innate sensitivity of most tumors (at least initially) to the crude and
blunt types of classical therapies we currently use. Clearly, such
innate apoptotic sensitivity is eroded during tumor evolution, and
especially in response to the strong selective pressure of classical
cancer therapies. However, such erosion is an evolutionary process and
arises through a specific and restricted repertoire of anti-apoptotic
mutations that the tumor acquires. Because of this, tumor cells remain
chronically dependent on their restricted repertoire of anti-apoptotic
mutations for their survival. In contrast, normal cells have no
pro-apoptotic oncogenic lesions and are supported by a variety of
trophic signals by virtue of their residing in their correct orthotopic
somatic environments. By definition, tumor cells are outside their
orthotopic environment and should therefore be acutely sensitive to
therapies that negate their anti-apoptotic mutations.

Unfortunately, we know too little as yet of the range or repertoire of
anti-apoptotic mutations that operate in different types of cancer cell.
Since such mutations are acquired through natural selection, and since
different selective pressures operate on different tissue types as they
evolve into tumors, anti-apoptotic mechanism are likely to vary between
different tumor types. This provides an unparalleled wealth of possible
therapeutic targets, if we could but identify them. This must remain a
priority.

**Conclusions**:

[Pathways]{.underline}

A substantial body of data exists on the pathways of responses to DNA
damage in bacteria and yeast, and studies of pathways generally follow
two main strategies: mutations in pathways that trigger cell death, and
responses of cells (i.e., responses at the cellular level) to damage.
Despite extensive information on some pathways in certain organisms,
many key gaps in knowledge exist in how cells detect and repair DNA
lesions and orchestrate these cellular responses. Further investigation
of DNA repair mechanisms in mitochondria and of the mechanisms of target
resistance and sensitivity is also warranted. We also need to increase
our knowledge of how DNA damage kills cells that lack a functional p53,
and the role of metabolism and microenvironment on cell death decisions
in response to DNA damage. We have accumulated clustered knowledge in
areas such as apoptosis and DNA repair, but we lack the knowledge needed
to link these clusters. The basic mechanisms of DNA repair and molecular
interactions in signal transduction pathways are probably best studied
in cellular models, including yeast and mammalian cells, and advances in
these areas may assist in the identification of novel therapeutic
interventions (e.g., IR and targets of NHEJ repair).

It is also critical in this area of investigation to understand the
pathophysiology of normal versus tumor tissue and the progression of
normal tissue to tumor tissue. It is assumed that mutations in
mechanisms that alter DNA repair or damage response pathways can
potentiate tumor development. However, it is not clear whether this
process is (1) a stochastic phenomenon that "opens the door" to a cell
progressing to transformation or to cell death; or (2) an ordered,
repeated process that continues until reaching a specific threshold
beyond which repair no longer occurs, ultimately generating a cancer
cell or causing a cell to die. Tracing the effect of a DNA repair defect
from its initial stage to the final outcome in a highly advanced tumor
cell would likely be highly informative in distinguishing between these
two possibilities and in identifying different death suppression
mechanisms that lead to variable sensitivity to DNA damage. Comparisons
of immediate responses to DNA damage, which appear to involve
posttranslational changes, and longer term adaptive responses, would
likely be informative.

Tumors may be considered as dysfunctional tissue. For example, tumor
cells are attracted to a type of vasculature to which normal cells are
not. In addition, specific lesions in tumors prevent terminal
differentiation. A more sophisticated analysis of signaling pathways
within currently curable human tumors would be enormously valuable.
While these tumors (mostly pediatric leukemias, sarcoma, germ cell
tumors, certain lymphomas) have been studied in the past, a more
sophisticated approach, which compares and contrasts their signaling
responses to chemotherapy (particularly DNA damaging agents) relative to
incurable tumors, is likely to shed important light on key variables in
treatment success.

[Tools]{.underline}:

Considering that the NCI Developmental Therapeutics Program is being
\"reshaped,\" the NCI could now play a direct role in providing
infrastructure and support for the development of new tools. The NCI
should be looking hard at data already in hand with an eye to making it
more accessible (e.g., cross-indices of chemical compounds vs. the
'NCI60' cell lines) or simply funding development to make needed
reagents available (e.g., screening to identify panels of
phospho-specific antibodies that work on tissue). A model is found with
the development of the PubMed database, where the previous MedLine was
rolled over into something that is web-accessible, readily searchable
and easily elaborated to add additional features at the local level.

In addition, improved, advanced computer and web-based systems modeling
programs relevant to DNA damage signaling also are needed. Participants
noted a two-dimensional computer model developed by Kurt Kohn that
incorporates the underlying biochemical mechanisms involved in the cell
cycle (a similar model for apoptosis also has been developed). Although
these models can be helpful and serve as a platform for further
investigation, participants generally agreed that a research model
should be dynamic and should allow for contingency pathways and multiple
decisions by the cell. It was noted that other more quantitative
computer models are under development. However, because of the limited
amount of data available, applications of these models currently are, in
turn, also limited.

Sophisticated mouse models that accurately recapitulate human neoplastic
processes are also needed. Most existing transgenic and knockout models
merely "mimic" cancer. Efforts, like that of the MMHCC, to more
faithfully recapitulate relevant neoplastic processes should be strongly
supported. Faithful mouse tumor models are essential since, as discussed
countless times in the Think Tank, the response of somatic cells (normal
and neoplastic) to DNA damage and/or other therapeutic insults is, in
great part, dictated by interaction between the tumor cell and its
dynamic somatic environment.

A particularly attractive suggestion regarding animal models was to
engineer \"reporter transgenics/knockins.\" Such mice would contain
germline-encoded reporters (such as GFP, LacZ, Luciferase) that would
respond to a variety of important signaling or transcriptional pathways
(eg p53 responsive, NFkB responsive, HIF, hypoxia, and many more). Until
molecular imaging becomes possible via PET (not likely for a while
still), such animals could prove enormously valuable.

[Human Tissues]{.underline}

Much of the emphasis in the past two decades had been on defining
genetic differences between tumor and normal cells. This has come, quite
naturally, from the emphasis on cancer as a somatic genetic disease
process. The most likely way to identify truly useful differences
between normal and tumor cells (i.e., those that when targeted would
have the offer the largest possible therapeutic indices) is to determine
which normal pathways in tumors are still functional and can be
exploited in the background of the tumor to force specific endpoints,
such as cell death. This analysis will be facilitated by the ability to
assay the activity of damage response networks by protein expression
levels.

Few tools are available to study responses to DNA repair in tissues.
However, such tools are necessary to map processes in normal and tumor
tissues. To accomplish this goal 'modern molecular pathology' needs to
be better defined in this context. For example, what functional imaging
or histopathology markers are useful to examine endpoints such as
apoptosis, energy state and viability in vivo or in tissue/tumor
specimens from humans or mouse models? Methods that cut across different
types of specimens: e.g., functional imaging, biopsy samples and
archival tumor specimens (currently the world's largest untapped human
tumor biology resource) would be particularly valuable. The suggestion
of phosphorylation site-specific antibodies that work on tissue is a
good example, as is simple staining for p53 abundance. This would be the
best place to fund some descriptive normal biology, which we desperately
need to provide the 'numerator' for all of the tumor studies and begin
to understand the tissue correlates of therapeutic index. Use of imaging
as a noninvasive tool to examine molecular pathways in carcinogenesis
and in response to DNA damage is still in the early stages of
development but holds great promise. Expanded use of skin (or biopsies)
was suggested as a model for imaging studies and assessment of the
pathology of normal versus carcinogenic cellular processes.

At least two types of effort are focused on mechanisms and markers of
DNA repair in human tumor tissue. They are academic-commercial
laboratory collaborations developing reagents (e.g.,
phosphoepitope-specific antibodies) and researchers conducting tumor
specimen analyses (e.g., chromatin status or modification in relation to
tumor type or stage). Forging better connections between the two would
facilitate movement of new analytical reagents and techniques into the
clinical setting and yield more information from human specimens.

Broader issues that need further investigation include gaining a better
understanding of the persistence of DNA damage versus transient DNA
damage systems; determining the relative rates of proliferation and
apoptosis in conjunction with DNA damage signals *in situ* (e.g., in
oncogenic mouse models); assessing processes in real time; conducting
longitudinal studies in humans; and understanding the different
mechanisms underlying chemotherapy and radiation. One notable gap that
warrants further investigation involves the distinct differences in the
*in vitro* versus *in vivo* responses to DNA damage and therapeutic
interventions (e.g., radiation). An approach to gaining a better
understanding of these differences and bridging the gap between these
systems may be through the study of pathway reporters inside tumors (see
Tools). Another general challenge to the field is the standardization of
technologies, assays, definitions, interventions, and outcomes across
research to improve the power of between-study comparisons.

[Targets, Strategies]{.underline}

The emerging science indicates that it will be possible to
differentially modulate sensitivity to DNA damaging agents in cancer and
normal cells. Practically all cancer cells have mutations targeting DNA
damage response genes. Mutations in these genes are essential for cancer
development and result in rewiring of the DNA damage response pathways
in cancer cells. Because of this difference in rewiring, inhibiting the
same molecule is expected to have different effects in normal and cancer
cells. We need to support research to identify and characterize DNA
double strand break sensors and transducers. These proteins are
promising targets for development of drugs that could specifically
enhance the efficacy of current cancer therapeutics. Research to
validate which of these sensors and transducers would be suitable
targets for development of cancer therapeutics should be supported,
including development of prototypical small chemical inhibitors to test
proof of principle. It is expected that the proposed research will
utilize a multitude of experimental systems, including human cells,
human cancer tissues, mouse models and lower eukaryotes, as each
experimental system has its own advantages and disadvantages and
knowledge gained in one system can be easily applied to the others. In
addition, tumor cells have different sensitivities to DNA damage and
other apoptotic stimuli. Sensitization models that focus on unique
mutations and/or unique clinical situations with high sensitivity may
help determine the role of DNA damage and repair in the carcinogenic
process.

It is critically important to explore ways in which sub-lethal
pro-apoptotic signals can be combined to overcome the
apoptotic-buffering threshold in cells. Merely refining, exacerbating or
increasing the persistence of DNA damage alone may not accomplish
selective tumor cell death. No amount of damage signaling is going to
provide a therapeutically discriminate pro-apoptotic signal if, as in
many tumors, DNA damage-induced apoptosis has become compromised by the
mutation of p53. Exacerbating DNA damage also carries with it the future
problem of inducing further oncogenic mutations. While it is reasonable
to argue that therapy-induced mutagenic/carcinogenic risk is secondary
to curing the immediate neoplastic disease in patients, it would be
better to avoid the problem completely if a better way of triggering
apoptosis in tumor cells can be found. No tumor cell has ever been
identified that lacks the apoptotic machinery if hit hard enough. This
reflects our understanding that the programmed cell death (PCD)
machinery is highly redundant and mechanistically defocused to employ
several overlapping processes, including mitochondrial dysfunction,
pro-phagocytic signaling, and caspase activation, each of which is alone
sufficient to ensure death of the affected cell. Therefore, it appears
that PCD cannot be lost by progressive mutation. Rather, tumors acquire
refractoriness to apoptosis through corruption of upstream signaling
pathways and through lesions that raise the threshold at which apoptosis
is triggered. Thus, adroit stimulation of multiple disparate apoptotic
pathways, each at a sub-lethal level, might together trigger activation
of the apoptotic program

Other research questions of interest focus on the role of the cell cycle
in DNA repair and cell responses to DNA damage. We must find out whether
blocking the cell cycle is a good clinical strategy.

**Specific Recommendations for the NCI:**

-   ***Analyses of the human DNA-Damage Response networks (DDR)*:**

    -   Complete the mapping of biochemical and regulatory circuitries
        that link the cell-cycle checkpoints, apoptotic and DNA-repair
        pathways into the hDDR with special emphasis on putative
        regulatory nodes.

    -   Develop a quantitative, computer-driven, systems-biology model
        for the hDDR network in human cells.

    -   Complete (or initiate) the integration of hDDR proteomics,
        gene-regulatory and gene expression databases with more dynamic
        cellular metabolite and energy profiles as functions of DNA
        damage.

-   ***Identification of Molecular Targets & Translational Strategies
    for Enhancing Programmed Cell Death over Cell Survival in Response
    to DNA damage:***

    -   Support more research on ways to modulate the
        activity/expression of early-time ("upstream") mediator and
        sensor proteins to activate the pathways of programmed cell
        death.

    -   Support more research on the underlying mechanisms of
        p53-*independent* programmed cell death, because in most tumors
        p53 is inactivated by mutations.

    -   Validate, as targets, mechanisms other than apoptosis that can
        induce cell death in human tissue, such as those linked to
        metabolic pathways that are altered in specific tumors.

    -   Place greater research emphasis on mechanisms that determine
        whether dual-function ("downstream") signaling proteins (e.g.,
        p53, E2F1) signal survival or death responses.

-   ***Normal versus Cancer Tissues:***

    -   Conduct comparative proteomic studies of tumor cells/tissues,
        progenitor cells, and normal cells/tissues to elucidate
        additional anti-apoptotic components altered in cancer.

    -   Characterize and catalogue molecules from the signaling,
        checkpoint and apoptotic parts of the hDDR network as possible
        therapeutic targets.

    -   Develop predictive profiles for radiation sensitive/resistant
        individuals; tumors with or without functional p53, ATM or other
        key signaling proteins, in order to tailor DNA-damage-based
        therapy to the patient.

-   ***Reagents to Study and Technology to Study the DDR in vivo**.*

    -   Develop a resource library of phospho-specific antibodies or
        other "readout" reagents that can provide quantitative measures
        of DDR signaling and output pathways (e.g., caspase induction
        and apoptosis; focus formation and DNA repair). This should
        include phospho-specific antibodies that can examine the
        activities of the DNA-damage signaling pathways in cultured
        cells and primary human tumors, before and after treatment and
        at different stages in tumor progression, etc.

    -   Develop small molecule inhibitors of specific targets implicated
        in the response of cells to radiation, particularly where the
        target protein three-dimensional structure is known.

    -   Promote dynamic imaging in the study of mechanisms of DNA repair
        in *in vivo* and *in vitro* studies.

    -   Develop programs or incentives to allow for research access to
        early-phase clinical drugs belonging to pharmaceutical companies
        and studies of pharmaceutical reagents in mouse models of human
        cancer.

    -   Support application of molecular imaging capabilities to monitor
        the dynamics and cellular compartmentalization of the hDDR.

    -   Convene a conference or think tank focused on new tissue markers
        and modern pathology to improve connections between
        investigators in basic science and clinical research.

**Executive Summary of Cancer Etiology Think Tank**

For many years, the focus in carcinogenesis has been on initiation, and
particularly on the direct induction of DNA damage by chemical
carcinogens. While this is an important aspect of carcinogenesis,
further progress depends on the use of a more balanced approach,
emphasizing that the carcinogenic process is continuous and dynamic.
Chemicals (both exogenous and endogenous) can contribute to tumor
formation at any part of the process, and do so by interacting with a
broad range of molecules including proteins, lipids and RNA, as well as
DNA. For example, studies investigating the pro-carcinogenic effects of
reactive oxygen and nitrogen species (ROS) (RNS) on cancer initiation
and progression identified a large number of cellular damage effects
along the progression continuum, including evasion of apoptosis,
insensitivity to antigrowth signals, self-sufficiency in growth signals,
limitless replicative potential, sustained angiogenesis, and tissue
invasion.

If a new, more comprehensive approach to carcinogenesis is to be
maximally productive, a systems biology approach will be needed to deal
with complexities head-on, focusing not on individual components but
rather on networks that can be measured, modeled, and manipulated. It
will be necessary to generate very large, highly accurate datasets
describing the behavior of all components in the system. This will
require highly sensitive, newly available technology that can identify a
range of biomarkers measuring exposure. Benefits that will flow from
these broadened investigations include:

-   The identification of biomarkers useful for assessing risk, for
    earlier diagnosis and for measuring therapeutic efficacy using newly
    developed technologies with extremely high sensitivity.

-   An understanding of how endogenous and exogenous chemical exposure
    impacts repair pathways, epigenetic changes, and protein and lipid
    function, and how they alter cancer susceptibility, which will lead
    to novel targets for prevention and therapy.

-   Development of better strategies for chemoprevention, exposure
    avoidance, and healthy lifestyles

The Think Tank identified knowledge gaps and resources needed to improve
prevention strategies and identify at-risk populations that can be
summarized into 5 broad areas:

-   **[Chemical processes and pathways]{.underline}:**  Expand studies
    to include a broader spectrum of the effects of endogenous/exogenous
    chemicals and their reactions in the carcinogenesis continuum.  Use
    a systems approach to interacting signaling networks at the cellular
    and microenvironment levels.

-   **[Biomarkers]{.underline}:** Design approaches for the development,
    validation and application of chemical biomarkers of exogenous and
    endogenous carcinogen exposure. Use damage products measurements
    (e.g., DNA, protein, and lipid changes; urinary and plasma
    metabolites) for early detection, risk assessment, and monitoring
    therapeutic efficacy.

-   **[Models]{.underline}: ** Develop models with sufficient dynamic
    range to study combined chemical exposures and enable modulation of
    endogenous chemical products via knockdown, pharmacologic,
    chemopreventive, or dietary manipulation. A range models are needed,
    from microbes through vertebrates to three-dimensional organ culture
    systems.

-   **[Technology:]{.underline}** Enable collaborative access to newly
    developed, high sensitivity, high resolution, expensive
    instrumentation for high-throughput data collections.

-   **[Recruitment, collaboration and resources:]{.underline}** Train
    the biologists, chemists and modelers who must work collaboratively
    on chemical carcinogenesis in an interdisciplinary environment.

**Introduction**

The classic Berenblum paradigm of multistage carcinogenesis,
conceptually dividing the process into discrete stages of initiation,
promotion and progression, requires extension and modification to take
into account current advances in cancer research. It is clear that
carcinogenesis is a continuous, dynamic process. Human cancers are
caused or modified by exogenous and endogenous chemicals, and the same
chemical can have multiple effects along the initiation to progression
continuum. To eliminate the burden of cancer, research priorities must
reflect the continuity and enormous complexity of the carcinogenesis
process. Although the identification of exogenous carcinogens is nearly
complete, the identification of endogenous carcinogens is not. In many
cases, exogenous exposure and endogenous processes predispose to cancer
through the same ultimate effectors, such as ROS and RNS. Similarly,
much is understood about metabolic activation and detoxification, about
DNA adducts, and DNA repair, but knowledge of the biological
consequences of DNA adducts is incomplete, and the effects of
carcinogens on other molecules, such as proteins and lipids, are largely
unknown. Critical to further advances, both animal and human studies
highlight the need to consider the timing and duration of chemical
exposure. To understand the contributions and interactions of all these
factors, a systems biology approach is essential.

Animal models suitable for studying chemical carcinogenesis are very
limited. While genetic loss-of-function mutations (largely knockout
mice) have provided extremely valuable insights into genetic factors in
tumor development, these ablative models often do not replicate the
effects of chemicals. For example, modulation (vs. ablation) of DNA
repair pathways results from exposure to endogenous and exogenous
chemicals. As a result, while most cancers do not exhibit mutations in
DNA repair pathways, damage continues to accumulate.

A theme that permeated the Think Tank and can be seen in much that
follows is the importance of having biologists and chemists work in
close collaboration to maximize progress in the field. Biologists are
familiar with the complex changes in cell biology and physiology that
occur during cancer development and progression, and have expertise with
*in vivo* experimentation. Chemists understand the reactive potential of
carcinogens, have the ability to synthesize proposed intermediates in
carcinogen activation, and have access to technology that can identify
carcinogens and their metabolites. Ensuring collaborations between these
groups is complicated by differences in scientific approach and, often,
their location in different schools within a university.

The Think Tank recommendations can be summarized under five topic areas:
I. Chemical processes and pathways; II. Biomarkers; III. Models; IV.
Technology; and V. Recruitment, Collaboration, and Resources.
Recommendations in each area are summarized below, followed by
discussion points from the presentations and the literature.

 **[I. Chemical processes and pathways]{.underline}:**  The field's
research focus must expand to include a broader spectrum of effects
resulting from exposure to endogenous and exogenous chemicals throughout
the carcinogenesis continuum.  Such investigations should include the
consequences of exposure on RNA, lipids, and proteins, and epigenetic
modifications to DNA and protein. To accomplish this, it will be
important to investigate the consequences of infection and tumor
promoter exposure on cancer development and to identify signaling
pathways that are either dependent or independent of reactive
oxygen/nitrogen species (ROS)/ (RNS) and other effectors of
inflammation. Interdisciplinary teams will use a systems approach to
determine how cells and signaling pathways interact within target cells
and in their tissue microenvironment in response to carcinogen exposure.
To learn how committed stem cells in target tissues respond to chemical
exposures and how such exposures modulate their function may be a key to
understanding their resistance to therapy and their persistence
following treatment-induced remission.

**A) Carcinogen effects on RNA, lipids, proteins and DNA:** DNA damage
and mutation occur along the continuum of carcinogenesis, not just
during 'the initiation phase'. The field has focused on DNA, but
carcinogens also damage proteins, RNA, and lipids. DNA, protein and
lipid damage recovery pathways intersect, and responses to protein and
lipid damage are just as important as DNA damage in mediating cell
recovery from exposure. Although protein damage has been understudied,
recent work identified protein adducts as biomarkers. For example, a
mouse skin carcinogenesis study detected 95% of the labeled carcinogen
bound in damaged protein and most of the remainder in RNA; the smallest
amount of the label was associated with DNA. Even less is known about
the consequences of carcinogens on lipid metabolism, or the pro- and
anti-apoptotic effects of peroxidized lipids. The role of bioactive
lipids in signal transduction needs attention and recent technologies
have now enabled an investigation of these processes. Other factors that
should be considered are:

> ***DNA Adducts*:** DNA adducts can lead to p53 gene mutations in
> specific tissues like bronchial epithelium. The p16 gene is only
> methylated among smokers. Neither the tissue specific responses to
> exposures nor their underlying mechanisms are understood and they
> require investigation.
>
> ***Pathways:*** When epigenetics, protein pathways, and apoptotic
> pathways, are considered, it seems it is not specific genes, but
> pathways that are consistently altered in carcinogenesis. To identify
> pathways that influence cell death and mutation, genomic phenotyping
> for damage sensitivity could be useful. It is important to keep in
> mind that more than 1000 yeast proteins are involved in recovery from
> some carcinogenic agents. Because several interacting pathways are
> involved in recovery from a carcinogenic insult, it is important to
> identify synergism among these recovery pathways.
>
> ***Timing of exposure:*** The timing of exposure over the life course
> can be critical with respect to an individual's risk of developing
> cancer. Further, huge threshold differences in metabolism and
> detoxification may relate to differential gene expression at specific
> developmental stages; these differences are not well understood.

**B) Role of diet, hormones and carcinogenic processes:** Effective
prevention requires the identification of exogenous and endogenous
carcinogens and their interactions. Among other environmental agents,
dietary substances may be a key factor. Dietary effects operate in a
background of genetic differences and circulating endogenous hormones,
both or either of which can alter an individual's exposure risk.

**C) The roles of inflammation, ROS and RNS and microbial flora:**
Inflammation is "the perfect storm" for carcinogenesis, causing DNA
damage and activating production of growth-stimulatory cytokines.
Anti-inflammatory compounds have chemopreventive effects in animal
models and humans. Recently, attention has turned to the role of
inflammation, including reactive oxygen species (ROS) and reactive
nitrogen species (RNS), throughout cancer initiation and progression.
ROS and RNS cause a variety of different types of cellular damage; in
addition to increased mutation rates, cellular damage results in evasion
of apoptosis, insensitivity to anti-growth signals, self-sufficiency in
growth signals, limitless replicative potential, sustained angiogenesis,
and tissue invasion---all hallmarks of tumorigenesis. Declining defense
mechanisms during aging may increase sensitivity to inflammation. A
number of human cancers result from the combination of infection and
carcinogen interactions. Infection contributes to inflammation, but
pathogenic processes specific to certain infectious agents also play
important roles in carcinogenesis. The synergy of the hepatitis B virus
(HBV) with aflatoxin is striking\--the occurrence of liver cancer
increases dramatically in infected people. The appropriate analysis of
these complex interactions requires a systems approach.

**D) The microenvironment in carcinogenesis:** Although only one cell
type may be capable of forming a given tumor, chemical exposure of the
microenvironment is likely to have direct and/or indirect consequences.
Exposure of animals to a carcinogen that directly affects one cell type
may indirectly influence other cells in the tissue environment. In terms
of prevention, inhibition of IKKβ in target cells is more effective than
its inhibition in the surrounding inflammatory cells. An analysis of the
different cell types' sensitivity may reveal chemical interactions in
specific genetic pathways.

**E) Tumor promotion:** If it is possible to distinguish between factors
affecting tumor initiation (genetic changes) and those that give the
target cell its proliferative advantage, chemoprevention strategies
might focus on inactivating the latter. Carcinogens not detected by the
Ames test are likely to be tumor promoters, and are likely to involve
ROS. Dioxin is an example of a substance that causes no DNA damage, but
is a potent promoter in human skin and liver, acting through a single
receptor that alters gene products influencing apoptosis, ROS, and
cytokines*.* Better assays to detect tumor promoters should be
developed. Age and gender also influence tumor promotion and are
relevant to exposure assessment. For example, the ability to remove
oxidative damage from the prostate may diminish with age and the
commonly observed loss of COX2 activity in prostate cancer. Age and
gender effects are clearly illustrated in diethylnitrosamine induction
of hepatocarcinoma.

**F) The analysis of complex carcinogenic mixtures:** The assessment of
complex mixtures of compounds, such as tobacco smoke, remains a
particularly challenging area that requires considerable attention and
resources. There is a scientific consensus that mixtures need to be
investigated, but methods do not exist to address the complexities
inherent in such studies at budget levels that will survive the NIH peer
review process. Until now, most bioassays have used single, large dose
exposures to a single chemical. Findings from the analysis of a single
adduct species in a clean system cannot be extrapolated to real life
exposures. People are much more likely to experience combinations of low
dose exposures. A consensus is needed on scientifically acceptable
methods to study mixtures in cells and animals that take into account
differences in susceptibility due to developmental stage, genetic
polymorphisms, and gender.

**[II. Biomarkers]{.underline}:** Although biomarkers were not initially
a part of the Carcinogenesis Think Tank agenda, chemists and
carcinogenesis specialists are especially well equipped to identify them
since biomarkers represent a spectrum of chemically complex substances
such as chiral lipids, and endogenous protein and DNA adducts.
Biomarkers can be used to monitor exposure and treatment, and as tools
for early diagnosis. The chemists\' focus can now be expanded to
detecting protein, lipid and DNA damage. Epidemiologists need biomarkers
of past exposure history. A key issue in animal studies is to identify
methods to serially sample the same animal using urine or plasma rather
than having to serially sacrifice animals. This would dramatically
reduce the number of animals needed in carcinogenesis experiments.

> **A) Assessment of past exposures:** Samples collected during
> epidemiology studies can be used to determine past exposure history of
> the subjects once carcinogen biomarkers are identified. Although hair
> is regularly used for chemical analysis, its use for protein
> assessment is questionable. Tools with sufficiently high sensitivity
> to study biomarkers reflective of low exposure levels are needed.
>
> **B) Detection of present disease:** Since tumors have different
> patterns of protein expression than normal tissues, it should be
> possible to identify altered protein patterns in blood or urine as
> cancer biomarkers via proteomics. Biomarker development and validation
> require both animal and human studies. Markers that track disease
> processes or predict cancer progression would be especially useful.
> The study of liver cancer in China illustrates that a successful
> biomarker study requires a high risk population so that sufficient
> numbers are available for statistically meaningful results. The study
> revealed a non-linear interaction of HBV and aflatoxin in determining
> liver cancer risk.
>
> **D) Prediction of future disease:** *Fortune* magazine suggested that
> "The NCI should commit itself to finding biomarkers that are
> predictive of cancer development." The development of DNA, protein
> and/or lipid biomarkers that indicate cancer potential is a major
> goal. They could provide targets for chemoprevention strategies and
> guide patient counseling on lifestyle choices. Biomarker
> identification teams should also identify mutations so that adducts
> can be correlated with key mutations. 

**[III. Models:]{.underline}** Discussion focused on animal models that
have the potential to provide a mechanistic understanding of
carcinogenesis. It is important to determine the appropriate model to
use, since a given model may be useful for a particular organ system,
but not for all. Two models illustrate available insights:

> ***Hepatocarcinogenesis:*** Rat liver hepatocarcinogenesis was induced
> by genotoxic hepatocarcinogens and the initiated preneoplastic cells
> were isolated. In wild-type animals treated with a single *in vivo*
> dose of the hepatocarcinogen diethylnitrosamine (DEN), superoxide
> production by the Kupffer cells increased and enhanced DNA damage and
> nitrotyrosine in liver proteins. Phox-/- knockout mice treated with
> DEN showed less DNA damage and almost no nitrotyrosine production. It
> thus appears that the cell injury, DNA damage, apoptosis/necrosis, and
> proliferation result principally from increased superoxide release by
> Kupffer cells. This model illustrates the importance of interactions
> between different cell types during the first steps of carcinogenesis.
> Infection, intestinal problems and ethanol can also stimulate Kupffer
> cells, and thus influence carcinogenesis.
>
> ***Genetically Modified Animals:*** When induction of NF-κB in mice is
> accompanied by deletion of IKKβ in intestinal epithelial cells, tumor
> incidence decreases. NF-κB, a transcription factor that regulates the
> expression of anti-apoptotic genes, decreases the susceptibility of
> cells to apoptosis, and may have a role in tumor promotion. In this
> model, IKKβ and NF-κB provide a molecular link between inflammation
> and cancer. However, in models of hepatocellular carcinoma, IKKβ
> disruption increases tumor number and size, as well as increasing
> apoptosis. In organs that can regenerate, increased apoptotic cell
> death pushes more cells into the proliferation cycle. Hence, a complex
> relationship exists between apoptosis and tumor formation, and
> promotion.

**A) Animal model**s**---strengths and weaknesses:** Animal studies,
particularly using rodents, have been the backbone of chemical
carcinogenesis research. New methods of genetic manipulation in these
species, and particularly in mice, offer promising new opportunities for
research. The NCI Mouse Models Consortium is a useful resource to the
carcinogenesis community. Some newer mouse mammary tumor models are
metastatic, resembling the human situation. Mouse models may prove
useful in identifying the carcinogens that cause breast, bone, and
prostate cancer. Interestingly, a mouse model that develops lung cancers
in response to cigarette smoke involves many of the same genes
associated with human lung cancers. Do these genes play a role in
initiation and/or progression? However, studies in rodents also have
limitations. For example, tests have revealed that only 40% of chemical
carcinogens harmful to animals are also harmful to humans, and at lower
doses, only half of these were toxic in humans. Most mouse tumors are
rarely metastatic or invasive, and mice do not develop gastric cancer.
Additionally, mice have higher glutathione transferase levels than do
humans, which limits their usefulness for some types of research. Gene
knock-out mice are widely used, but there is some concern that the
complete absence of a gene is not always a good mimic of a drastically
reduced level of expression of the gene, a situation more commonly seen
in carcinogenesis. Aflatoxin studies illustrate another limitation using
selected animal models. Fisher 344 rats metabolize aflatoxin much as
people do, but cannot be infected with hepatitis B virus (HBV). Thus,
there is no animal model that truly replicates the interaction of HBV
and aflatoxin observed in human studies, but one should be developed. In
looking for models beyond rats or mice, what are the options? Lower
eukaryotes can be useful, particularly where the specific mechanisms
involved are known. In DNA repair, the choices are yeast, mammals, or
*C. elegans*; little is known about DNA repair in *Drosophila.* The
growing appreciation of the importance of stem cells in carcinogenesis
suggests that *in vitro* studies of embryonic and adult stem cell
cultures may be very useful. Currently, methods to maintain and
manipulate such cultures are limited, but rapid progress is anticipated.

**B) Inflammation is a confounding factor in animal studies:** Even
brief inflammatory episodes during the course of a carcinogenesis study
can affect the outcome. The flora in animal facilities differ, so the
occurrence of tumors may be high in one facility and low in another. The
existence of at least 20 types of *H. pylori*, makes it possible to miss
their presence in supposedly *H. pylori*-free animals. If the
inflammatory agents (pathogens) are removed, the ability to generate the
phenotype (tumors) may be lost. Despite its importance, few animal
models are available to study inflammation.

**[IV. Technology]{.underline}:** Think Tank discussions of technology
focused on four broad areas: A) new technologies, B) shortcomings of
techniques, C) sensitivity and other challenges, and D) fiscal
constraints.

**A) New technologies**: Mass spectrometry has been the most widely used
analytical technique in chemical carcinogenesis, and spectacular strides
have been made in this area in recent years. Real-time mass spectrometry
with blood flow detection is a reality. Various types of mass
spectrometry have improved to the point that they are approaching their
limit of sensitivity. New types of mass spectrometers include the
exquisitely sensitive Fourier transform ion cyclotron resonance (FTICR);
MALDI-TOF/TOF, which can characterize proteomes, lipidomes, and DNA
adducts; and the high resolution triple quadrupole, which can quantitate
lipids, DNA adducts, proteins and protein adducts. Current work involves
increasing their specificity still further.

Proteomics, lipidomics, and related genomic-level, high-throughput
analyses are needed in carcinogenesis, as they are in many other areas
of cancer research. Mass spectrometry is a very useful technique in
these areas, but more technologies are needed. Further techniques of
protein analysis utilize radioactive labels to detect and pinpoint
changes in protein patterns after a challenge. Stable isotope proteomes
can be used as standards to run in 2-D gels. The proteomics laboratory
at the University of South Carolina is using chromatography columns on a
chip, which are more sensitive than existing separation techniques.
These chips should be generally available in two to three years.

The importance of non-invasive analytical procedures has been emphasized
above, and intravital microscopy offers substantial promise in this
area. Two-photon microscopy can penetrate tissue, at least to millimeter
levels. It is possible with this technology to measure activities
spectroscopically, an ability which could be applied to look for a ROS
spectral signature.

**B) Shortcomings of techniques:** There are many areas in which
technological improvements are still needed. Inferring sequential
changes in animals from serial sacrifice has serious pitfalls. The
development of non-invasive techniques to track changes in a single
organism would substantially improve the quality of such data. In the
case of an inflammatory response, for example, an adequate imaging
methodology to obtain real-time records of organ-by-organ changes would
be invaluable. 2-D gel analysis and protease digest proteomics are not
quantitative. Even using antibody array information to detect
up-regulation of proteins may fail, if it is applied at the wrong time,
and information on labile protein modifications is very easy to lose
during analysis.

**C) Sensitivity and other challenges:** Analytical and instrumental
sensitivity was a recurring topic of concern. More sensitive mutagenesis
assays are needed\--current assays cannot detect mutations less frequent
than one part in 10^6^. For detection of many endogenous substances
(such as endogenous vinyl chloride at the DNA and gene level), more
sensitive and artifact-free instrumentation is required. Assessing the
carcinogenic potential of low--dose exposures is difficult, as is
interpreting the "U"-shaped curves which may result. Ongoing challenges
for technology include methods to separate modified from unmodified
adducts; the tools to do a mass balance experiment, looking at many
pathways in a system such as chlorination and bromination; the chemical
means to detect changes in real time, perhaps using *in vivo* probes;
improvements in the chemistry of detection so that not only end products
of a reaction are detected; and the means to link carcinogens clearly to
disease or disease subtypes.

**D) Fiscal constraints:** Some sensitive instruments are costly to
obtain and maintain. Researchers are slow to invest in technology not
available within a P01 or RO1 budget structure, until its suitability to
their research challenges has been well-established. As a result, MRI or
PET scans are rarely done in animal facilities due to their expense,
although MRI resolution can show oxidative stress levels, changes which
might have regressed by the time animals were euthanized.

**[V. Recruitment, Collaboration, and Resources]{.underline}:** Although
not among the designated discussion topics, personnel and resources were
regularly mentioned as factors impacting the success of research
programs and the future development of the field.

**A) Recruitment:** Students perceive the field of chemical
carcinogenesis as doing the same thing for the past 30 years\--testing
chemicals and not asking mechanistic questions. Academic structures can
be blocks to cross training, although some institutions have integrated
chemistry with biology or instituted interdisciplinary programs that
expose students to research in carcinogenesis. T32 grants to support
young people entering the field and multi-disciplinary training grants
funded by NCI have been effective in training and recruiting new people,
but more effort is needed in this area. Think Tank participants agreed
that a combination of improved marketing approaches and funding can
attract promising graduate and post-doctoral students for future
leadership in the field of chemical carcinogenesis.

**B) Consortia:** Consortium grants, including those within a single
institution, were attractive to some Think Tank participants. Sharing
biological specimens, tissue arrays, databases, and other resources
benefits the research community and should be required. NCI can assist
by coordinating efforts to acquire and divide up tissue (especially
human samples), and provide material to investigators as needed.
Collaboration between chemists and biologists is also productive, and is
relevant to the recruiting concerns voiced above. In the context of the
roadmap initiative, some participants had done an exercise regarding
inter-and trans-disciplinary research and how to form a team. The
essential factor seemed to be that everyone must address the same,
specific question. Other Think Tank participants expressed considerable
skepticism about the benefits of consortia. Some advised caution in
forming large groups across the country, which can be inefficient in
getting information and publications together. In one opinion, the worst
thing NCI could do is to have a big urine bank or blood
bank\--researchers should be closely associated with the collection
process in order to know the source and storage conditions.

**C)** **The Grant System: A** major impediment to consortium formation
is the lack of convenient funding mechanisms. NCI's strict rules for
funding a program projects were viewed as counterproductive. One
solution is for NCI to develop the needed infrastructure by funding
individuals to develop these resources. Some believe there is a lack of
knowledgeable study sections members. People with seniority and
collaborative experience aren't on these sections, and conflict of
interest requirements preclude participation by the most informed and
up-to-date people because of their previous associations or
collaborations.

**Specific Recommendations for the NCI:**

The Think Tank identified knowledge gaps and needed resources to improve
prevention strategies and identify at risk populations. Technologies
that expand discovery opportunities are now available.

**Chemical processes and pathways:**  

-   Expand research focus from DNA adducts and mutation analysis to
    include a broader spectrum damage resulting from exposure to
    carcinogens throughout the continuum of tumorigenesis. 

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-   Develop a systems approach to interacting signaling networks at the
    cellular, microenvironmental and macro-environmental levels.

-   Expand studies of reactive oxygen species and other mediators of
    inflammation to identify the variety of cellular and
    microenvironment damage they cause.

-   Enable studies of low levels of exposure, in which perhaps 1 cell in
    1000 is altered. DOE support of studies to look at the effects of
    low levels of radiation that cause no change in cell cultures could
    serve as a model.

-   Identify carcinogens and other etiologic factors in breast, colon,
    and prostate cancer.

**Biomarkers:**  

-   Provide resources for the development, validation and application of
    chemical biomarkers resulting from exogenous and endogenous
    carcinogen exposure and their damage products (e.g., DNA, protein,
    and lipid changes; urinary and plasma metabolites)

-   Identify biomarkers for early detection, risk assessment, and
    monitoring therapeutic efficacy; identify markers that track past
    exposure to carcinogens.

-   Develop instrumentation for high-throughput sample processing, to
    permit multiplexing analytical procedures for biomarkers.

**Models:**

-   Develop models with sufficient dynamic range to permit analysis of
    combined chemical exposures; enable modulation of endogenous
    chemical products via knockdown, pharmacologic, chemopreventive, or
    dietary manipulation.

-   Provide more realistic funding for studies using genetically
    modified mice. In many cases, investigators must produce 3-4 times
    the required number of animals to obtain the desired genotypes. When
    modifier genes are found, follow-up studies are often not done
    because they can take three years and 1000 cages of mice.

-   Make effort to standardize animal nutrition, health status and
    knowledge of endogenous bacterial flora. Encourage studies in which
    all investigators use animals with the same microbial flora.

-   Use models of cancer progression, as well as initiation and
    promotion, for studies of combined etiological agents including
    mixtures. Inflammatory processes must be considered when designing
    cancer biology models and in implementing human translational
    studies.

-   Develop a useful spectrum of biological systems, including single
    cell-microbes, invertebrates such as *C. elegans* and *Drosophila*,
    vertebrates including rodents, and three-dimensional organ culture
    systems, which can be used to look at cell/cell interactions and to
    study tissue interactions.

-   Pay more attention to timing of exposure and the influence of
    developmental stages on carcinogenesis.

-   Develop a controlled carcinogen-induced tumor model in which
    proteomics and genomics can be used to look for chemical/biological
    interactions.

**Technology:**

-   Using available, newly developed high sensitivity, high resolution
    instrumentation, develop high throughput technology in **cooperative
    arrangements**. Due to their expense, requirement for high level
    expertise, and the need for quality control, such instrumentation
    must be shared.

-   Establish and support instrumentation centers within major
    institutions to make costly technology widely available.

-   Develop non-invasive technology (e.g. imaging and serum sampling) to
    track changes over time, (avoiding serial sacrifice); develop
    methods to do mass balance experiments, improve the chemistry to
    detect changes in real time (in vivo probes). Develop technology to
    mark tumor stem cells and initiated cells for visualization and
    isolation.

-   Design approaches to make stronger *in vivo* correlations between
    adducts, biomarkers, mutations, initiated and pre-neoplastic cells
    and cancer---a process limited, at least in part, by our lack of
    high-sensitivity mutational assays to extend dose-response curves
    for biomarker-mutation correlations.

**Recruitment, collaboration and resources:**

-   Support fundamental training in chemistry.

-   Support database development (an area currently buried in the depths
    of grant proposals) and mathematical modeling predictions of how
    biological systems respond to carcinogen exposure.

-   Increase support for developmental projects. These are currently
    ignored by NIH.

-   Support cross-training to emphasize the special role of chemists at
    all levels, including training grants and PI visits to collaborating
    laboratories

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-   Maintain NIH sponsored synthesis of high quality standards (labeled
    and unlabeled, small molecule and macromolecular).

**Executive Summary of the Epigenetic Mechanisms in Cancer Think Tank**

The purpose of the Epigenetics Think Tank was to bring together experts
from basic and translational sciences to discuss the potential of
epigenetic research to inform cancer biology, diagnostics and
therapeutics, and to make specific recommendations to NCI for action in
this field. The think tank began with brief presentations by each
participant on the first evening. On the second day, three discussion
sessions addressed cancer epigenetics, basic mechanisms in chromatin
structure, and epigenetic technology and epigenomics. A special
presentation was included on the European Epigenomic Network of
Excellence. On the final morning, the panel summarized the discussions
and prioritized recommendations.

The Think Tank highlighted a clear link between epigenetic alterations
and cancer. Epigenetic changes such as DNA methylation and imprinting
are targets of environmental carcinogens and diet, and probably are
responsible for much of the population variation relevant to cancer
risk. The role of hypomethylation in gene activation, aging and
chromosomal instability is an emerging significant issue. The panel was
particularly excited by the apparent role of epigenetic changes in the
earliest stages of carcinogenesis, because these are potentially
reversible with treatment. This provides an epigenetics-based strategy
for cancer prevention. Epigenetic changes also clearly play an important
role in tumor progression, tumor cell heterogeneity and possibly
metastasis. A key need is to map these early changes through the
analysis of methylation and other epigenetic marks in a well-defined set
of reference human normal and tumor tissues. In addition, it is
important to determine the temporal relationship of epigenetic
alterations, as well as their relationship to underlying DNA sequence
variation in the population.

This is an exciting time in the understanding of the mechanisms of
epigenetic transcriptional regulation and chromatin structure. Protein
complexes that manipulate nucleosomes, organize larger chromatin domains
and set boundaries of chromatin structure have been discovered in recent
years; now research must focus on discovering how they work in such
cellular functions as replication, transcription and differentiation.
Crucial next steps are to understand the role of key histone
modifications, cis-acting elements, and regulatory proteins in setting,
maintaining, and reprogramming epigenetic memory. Other key unknowns are
the determinants of higher-order chromatin structure, the function of
micro RNAs or siRNA in mammalian gene regulation and genome stability,
and the mechanism of transmission of epigenetic marks through cell
replication.

The Think Tank concluded that the highest priority should be development
of a U.S. Human Epigenome Project of analogous scope to the Genome
Project. The immediate establishment of a Working Group to define the
criteria and plan the approach to the Epigenome Project is timely and
highly recommended. There was strong consensus that we are now at the
stage at which we can \"genomicize\" epigenetic information. Most of the
necessary technologies are already available, although some tools for
analysis of epigenetic alterations at the genome-wide level are still
needed. New quantitative and computational methods may be required,
including integrating epigenetic information with existing databases.
Mapping epigenetic information to the human genome will maximize its
usefulness and allow us to determine the relationship between genetic
and epigenetic variation in the population, and its relationship to
cancer. Such a project would be immediately worthwhile for its basic
science insights as well as for enabling the use of epigenetic
information in prevention or therapeutic strategies.

**Introduction**

The think tank began on the first evening with brief presentations of
scientific advances, questions and barriers by each participant. The
following day, three discussion sessions addressed Human Cancer
Epigenetics; Basic Mechanisms in Chromatin Structure and Epigenomics;
and Technology and Translation. There was also a presentation on the
European Epigenome Program. On the final morning, the discussions were
summarized, and the participants created a prioritized list of major and
minor recommendations to NCI which will best advance the science.

In each session, the discussion was organized around a set of major
questions. The panel began with these questions, and proceeded to
discuss the issues and most critical next steps, and concluded each
discussion with the next crucial questions to be answered and needs for
the field to progress.

**Human Cancer Epigenetics**

*Mechanisms of cancer initiation and progression by epigenetic events*

Epigenetic changes are modifications to the genome that are heritable
during cell division but do not involve change in DNA sequence.
Expression of individual genes, of gene regions or a whole
transcriptional program are regulated not by the DNA sequence, which is
the same in every cell, but by the epigenetic marking and packaging that
regulates chromatin structure. DNA methylation, histone variants and
post-translational modifications, nucleosome positioning factors,
boundary setting elements, and chromatin loop and domain organizer
complexes are all elements of the epigenome.

Alterations in methylation, imprinting and chromatin are ubiquitous in
cancer. A clear link between epigenetic changes and cancer, as well as
other diseases, has in many case been established. And beyond these
specific instances, epigenetics has the potential to explain many
aspects of cancer: these marks can be modified by environmental factors;
the age dependance of cancer corresponds to epigenetic changes in aging;
and the quantitative nature of epigenetic changes corresponds to changes
seen in cancer. The frequency of epigenetic alterations is orders of
magnitude greater than that of genetic mutation, but unlike mutation,
they are reversible.

The most frequently identified epigenetic alterations in cancer are
changes in DNA methylation, although others have been identified as
well. DNA methylation at CpG sequences (frequently in stretches called
"islands") is a basic marker of the genome that appears to have several
functions. Methylation of gene regulatory regions has long been
associated with transcriptional inactivation, and differences in
methylation pattern in the promoter regions of tumor suppressor genes
are documented in some cancers (e.g., p16/INK4a in pancreatic cancer).
Hypomethylation is thought to either permit gene expression where it
should not occur, or to encourage chromosome instability, especially
when it occurs in the centrosome region. In general, global
hypomethylation with local hypermethylation of CpG sequences is
associated with cancer, although this concept may eventually be revised.

Two well described instances of epigenetic changes involved in cancer
were used to illustrate different epigenetic mechanisms of cancer
initiation throughout the meeting. Both involve loss of imprinting
(LOI), which is DNA methylation that silences specific genes on either
the maternal or paternal allele lineage. In Wilms' tumor, approximately
95% of cases do not follow the two-hit model as it has been understood,
with an inherited mutation in one allele of a tumor suppressor followed
by a mutation in the other allele; instead, the second hit usually is
LOI that activates the IGF2 gene. In colon cancer, IGF2 is also
frequently activated, but another mechanism is involved, in which
genetic and epigenetic mechanisms appear to be complementary. The LOI
seen in these tumors also exists in the surrounding normal tissue and
appears to predate the tumor; the theory is that this "field effect"
renders the mucosa highly susceptible to subsequent insults, such that
the overexpression of IGF2 (or other growth factors) due to LOI renders
mutations such as APC mitogenic rather than apoptotic.

While the link between epigenetic changes and cancer is clear, the
mechanisms behind the abnormal changes -- indeed, the mechanisms by
which normal patterns are established and maintained -- at this time are
largely unknown. The relationship between cause and effect is also
controversial; while histone acetylation and DNA methylation are
permissive for transcription, there is conflicting evidence as to
whether these marks are the signals for gene expression, or if they
follow the passage of the polymerase and serve to reinforce the "active"
gene structure. DNA methylation differences at specific loci exist in
Wilms' tumor and Beckwith-Wiedemann syndrome, but a mosaic of
methylation states is observed in the population of tumor cells. This
raises the question of the degree to which the methylation pattern is a
cause of the malignant growth. Because normal methylation patterns are
not well defined, it is difficult to determine what is abnormal in
cancer. In spite of the widely documented link between methylation
changes and cancer, none of the known methyltransferase genes are known
oncogenes or tumor suppressors. Thus, several crucial questions remain
to be answered for methylation to be optimally used in cancer diagnosis
or treatment.

A fascinating current issue is whether there are sequence variations in
the human genome that create susceptibility differences by predisposing
the site to epigenetic changes. Noncoding DNA such as repetitive
elements and transposons may play a role in genome-wide methylation
patterns. These elements are the main sites of CpG methylation in the
genome. The example discussed by one think tank participant was that of
a transposed element that altered the methylation pattern at a gene
locus in a mouse model in a manner that responded to dietary factors. In
the offspring, the transposon could be differently methylated depending
on the maternal diet, with a higher methylation state leading to an
obesity phenotype, demonstrating a means by which disease states could
have an underlying genetic/epigenetic basis that responds to
environmental factors. In this example, a genetic event led to the
possibility of stochastic epigenetic changes. There is currently no
known human locus where methylation variation leads to trait or
susceptibility variation, or where a transposable element creates a site
of potential expression variation; however, the likelihood was thought
to be high that such examples will be found.

Histone modification, especially acetylation, has long been recognized
to influence transcriptional capacity, but we must now begin to
understand how DNA methylation and histone modifications have
coordinated effects. A self-reinforcing cycle between these two
epigenetic marks appears to exist, such that transcriptionally active or
inactive regions of chromatin (i.e., euchromatin or heterochromatin) are
self perpetuating through replication. The most important next step will
be to understand the degree to which changes in these epigenetic marks
are the causative regulatory factor in chromatin function changes, or
whether other complexes play the regulatory role and these marks play a
mainly reinforcing or maintenance role. This issue was the focus of some
debate, since different results have shown these marks to play both an
active and a passive role. Histone acetylation, for example, has been
shown to cause transcriptional activity and also to result from the
passage of the polymerase complex, thus being self-reinforcing, while
loss of single specific histone acetylations can cause the spread of
heterochromatin over a gene region. The recently proposed histone "code"
of modifications was challenged by some panel members because such
questions of cause and effect cannot yet be answered.

While DNA methylation of specific genes or general pattern changes are
the most widely reported epigenetic signature in cancer, a cancer
connection with chromatin organization is also well documented. SNF5, a
subunit of the SWI/SNF chromatin remodeling complex family, is deleted
in the aggressive pediatric cancer rhabdosarcoma. It acts as a tumor
suppressor, activating expression of p16/INK4a and repressing several
cyclins and CD44, a cell surface glycoprotein implicated in metastasis.
Another example is that of the tissue-specific BUR (base unwinding
region)-binding protein SATB1, which appears to organize chromatin loop
domains and act as a positioning factor that attaches genes to Matrix
Attachment Regions (MARs), giving them the proper nuclear localization
for transcriptional activation. SATB1 specifically binds to the major
break region in the characteristic chromosomal translocation of some
lymphomas, creating the MAR which is thought to be a susceptible site.
It will be interesting to search for similar proteins specific to other
cell types and determine their expression or activity in cancer cells,
since SATB1 has the ability to cause global changes in gene expression
pattern.

Several specific needs were identified to advance our understanding of
the epigenetic mechanisms functioning in cancer. It will be important to
define genetic-epigenetic relationships; e.g., to identify mutations
that lead to epigenetic changes. It will also be necessary to determine
the relationships among such transacting modifiers of the epigenome as
methyltransferases, transcription factors and genome organizers, as well
as identify the factors and/or pathways that are activated in
conjunction with these relationships. Another significant area in need
of investigation is the effects and dynamics of epigenetic modifications
of repetitive elements, transposons, pseudogenes, and novel transcripts.
New insight into the organization and functioning of transcriptional
networks and pathways may be gained from understanding principles of
chromatin organization. New experimental model systems (e.g., to study
RNAi; global CpG methylation and hypomethylation in cancer; pseudogenes
in heterochromatin origins; and histone acetylation/deacetylation) are
needed to study these features. Finally, it will be important to
identify and develop applications for reagents for site-specific
modifications of histones, such as acetylation-site-specific antibodies,
and identify ways to decrease cost and increase availability of these
reagents.

*Interplay of epigenetics and cancer B where does epigenetics fit in the
big picture of initiation and progression?*

Despite the cancer associations highlighted above, the question remains
of how frequently epigenetic gene activation or silencing is a "first
cause" event that initiates dysplasia or creates susceptibility.
Depending on the cancer, the first hit may be a mutation followed by
epigenetic changes leading to a functional second hit, or abnormal or
age-related epigenetic changes may create conditions that enhance
environmental assaults or release suppression of tumor progression. Once
the primary tumor is under way, the many steps by which a motile,
metastatic phenotype emerges are likely to include epigenetic changes in
gene regulation. Tumor cells generally must obtain a motile, spindle
phenotype while traveling to the new site, then change phenotype again
as they become established as a metastatic lesion. Regarded in this
light, the reversibility and changeability of epigenetic states, as
opposed to mutations, is a thought provoking idea. The reexpression of
genes in metastatic cells, and the regulation of some genes involved in
the angiogenic switch, are thought potentially to have an epigenetic
etiology. In all of these questions, the lack of experimental models is
a major barrier. Specifically lacking are models of very early
epigenetic changes or very early stages of disease.

One promising focus for research is the question of epigenetic
methylation changes as a timing event in the path from stem cells to
differentiated cells to tumor cells (i.e., DNA methylation as a
developmental timer). Another area of investigation might involve
examination of epigenetic changes in "normal" cells and cells
predisposed to cancer, to distinguish between normal epigenetic
variation or changes with age, and potentially dysregulating epigenetic
changes leading to disease. The epigenetic status of normal cells
surrounding tumors, including angiogenic, immune, and stromal cells, may
also play a significant role, and is virtually unknown. It will also be
necessary to identify the relationship of epigenetic changes to
chromosome instability and recombination in cancer.

*Comprehensiveness of epigenetic studies of cancer B how much
information do we need?*

It will be important to conduct a genome-scale analysis of domains of
epigenetic change at the local level, in gene groups or clusters, and
globally. A genome-wide screen for DNA methylation changes would avoid
the bias from candidate gene studies; on the other hand, the data set
from a single region would allow us to study all epigenetic features,
and determine how they relate to each other. There were conflicting
opinions on which approach would be most valuable at this time. It will
be necessary to investigate the functional significance of specific
epigenetic marks in order to better understand how to resolve this
question. It is important to determine the relationship between
hypomethylation and gene activation, as there are several examples of
loss of methylation linked to new gene expression and cancer. Before
demethylating agents become widely used to treat cancers, this question
must be addressed. The relationship of epigenetic changes to histone
modifications is also important, and may involve the results of clinical
applications, such as the effects of drugs on gene expression.

It will be highly informative to add epigenetic information to existing
databases of genetic information. Gene regions could be layered with
information on methylation and other marks that relate to
transcriptional activity. However, as discussed in the final section on
Epigenomics, a judicious approach to selecting experimental material and
identifying the regions of chromatin to analyze must be used. To date,
drugs that target DNA methylation and histone acetylation have been used
with some degree of success in the clinic, albeit the more that is
learned about the function of these epigenetic marks the less clear it
becomes how the drugs work.

**Basic Mechanisms in Chromatin Structure**

*Key Players in Chromatin Organization*

A number of molecules with critical roles in chromatin organization,
remodeling and epigenetic modification have been identified in recent
years. However, our understanding of which ones are the key regulators
and how they accomplish their roles in development, in the identity of
different tissues, or during cellular functions such as replication is
far from complete. Further basic research to better understand the
mechanisms of action and determine the regulatory pathways of the known
enzymes and complexes is crucial and timely. At the same time, it is
important to try to broaden the spectrum of known chromatin modifying
enzymes, if we wish to form a comprehensive picture. To understand how
chromatin structure marked by specific epigenetic modification is
established, it is necessary to identify additional players at the
levels of DNA, RNA and proteins, and then go on to identify the dynamic
relationships between them. The role of non-coding RNAs is addressed in
a later section.

There is evidence that DNA methylation/demethylation affects gene
transcription by signaling chromatin structure changes, rather than
simply "blocking" access by polymerases or transcription factors. Thus,
since DNA methylation status is the most widely and readily measured
epigenetic mark, it will be important to determine its role in chromatin
organization. At the same time, it is important to search for long-term
epigenetic marks other than DNA methylation and histone modification.

A major challenge in the near future will be to identify key
determinants of locus-specific chromatin structures; that is, key
histone modification sites and key cis-acting DNA elements (e.g., BURs,
which are recognized by global gene regulators) which have dominant
roles in the formation of specific chromatin structure, as well as the
protein complexes which interact with these sites. To understand how
epigenetic states are established, it will be necessary to identify new
interacting proteins that are involved in chromatin targeting and
remodeling. These partner proteins currently include PCNA, RNA
polymerase, nucleosome assembly factors, CTCF, and loop organizers.
Other factors must be presumed to exist which have yet to be identified;
however, it is not too soon to begin to ask which part of the genome is
regulated by which of the known factors.

The known molecules that regulate accessibility of the DNA to regulatory
factors include DNA methyltransferases, histone variants, and enzymes
that covalently modify histones through acetylation, phosphorylation,
methylation and other chemical moieties. Another class of
chromatin-modifying complex includes those that move nucleosome
positions to reveal or occlude DNA sequence. Three families of these
complexes have been identified -- SWI/SNF, ISW1, and NuRD -- all of
which consist of a core subunit with an ATPase domain, plus associated
subunits presumed to be regulatory. Other chromatin modifiers include
the Methyl-CpG-Binding Domain (MBD) protein family, Position-effect
variegation (PEV)-related factors, and chromatin loop organizers.
Although presumed to exist based on the observed dynamics, neither DNA
demethylases nor histone demethylases have yet been identified.
Discovery of such crucial enzymes will have high impact on understanding
of chromatin structure maintenance and remodeling. Additionally, any of
these enzymes, loop organizers or boundary element-binding proteins may
be subject to post-translational modifications that regulate their
functions, although very little is known at all about such regulation of
these molecules. Many of these proteins can be studied in yeast; for
example, yeast studies have demonstrated that Sir2 histone deacetylase
induces the spread of heterochromatin, while Sas2 acetyltransferase
disrupts heterochromatin, and together, they appear to establish
chromatin boundaries. Efforts are now being directed at understanding
the roles of known higher eukaryote analogs of these enzymes.

*Mechanisms for higher-order folding of chromatin*

To understand chromatin structure and function, it is essential to
expand our knowledge of how chromatin is packaged beyond the nucleosome
structure. Which proteins are responsible for higher-order packaging
(e.g., loop domain organization) and the biological significance of such
chromatin folding are fundamental questions that have only recently been
formally addressed. It will be an important step to identify more loop
organizers in addition to SATB1, in different tissues and at different
developmental stages, and to distinguish between general and specific
proteins. This will require improvement in the highly variable loop
capture assay, and the development of new technologies to analyze
higher-order chromatin structures (e.g., in mega-base domains).

It is necessary to determine the role of loop structures in delineating
functional chromatin domains that are marked by specific epigenetic
modifications. Such an approach, which introduces the third dimension
into epigenetic analysis, will reveal the contribution of higher-order
folding to epigenetic modification and overall genome organization. It
will be important to identify the mechanisms that regulate functions of
loop organizers such as SATB1, and to discover additional analogous
protein complexes. Finally, it will also be important to analyze the
roles of loop structure and loop organizers in cancer and explore their
diagnostic utility. Some observations suggest that the organizer protein
BORIS competes with the related protein CTCF to disrupt boundary
function, and may alter transcription pattern on a large scale when
abnormally expressed in cancer. Additionally, the global organizer SATB1
is aberrantly expressed in some cancers. It will be significant to
explore whether these proteins could be effectively targeted in cancer
therapy.

*The role of RNA in gene regulation and genomic organization*

Non-coding RNA appears to play several roles in chromatin regulation.
Emerging evidence suggests that micro RNAs (miRNA) or siRNAs can
regulate over very large chromatin territories. These small RNAs,
produced by defined processing pathways, are known to silence genes at
the translational and perhaps also the transcriptional level; however, a
role in maintaining heterochromatic regions is currently only
postulated. It will be important to determine the generality of RNAi
control in the genome beyond repetitive elements, and the function of
RNAi in genome organization. Currently it is unknown how many miRNAs are
present in our genome. It is still to be determined how they can be
identified, and how their targets can be validated. Then, moving beyond
quantification of miRNAs, we will have to determine the role of miRNA in
gene expression, where it acts, and what link if any it has to cancer.
Another intriguing question is whether viruses and other pathogens
modify the epigenetic profile of the genome in ways that involve siRNAs
and/or miRNA, leading to dysregulation and potentially diseases such as
cancer.

Intergenic transcription is a novel mechanism by which heterochromatin
may be excluded from transcriptionally active regions. It will be
necessary to determine the link between intergenic transcription and
RNAi. An important question is whether siRNA-based regulatory networks
exist, and what the role and mechanisms of large noncoding RNA
transcripts (e.g., Xist) are in chromatin function.

*Epigenetic inheritance in somatic cells*

This area of chromatin epigenetics research in particular is still in
its early stages and warrants further, extensive investigation. DNA
methylation is initiated de novo by DNA methyltransferases and
maintained by Dnmt1, and this provides a mechanism of epigenetic
inheritance. However, there are epigenetic phenomena in organisms such
as *Drosophila* that lack any significant DNA methylation. Epigenetic
states may be transmitted through specific chromatin structures, such as
histone modifications, but how this is achieved is largely an enigma.
Lysine methylation on histones 3 and 4 provides relatively stable marks
on histones. But, again, how such marks are maintained through the cell
cycle or through development is not known, given that there are
mechanisms for active loss of histone methylation, in addition to
passive loss by dilution at each round of replication. Replication
timing may be a crucial mechanism in setting or preserving the
epigenetic marks, since euchromatin replicates early in the cell cycle,
while heterochromatin replication is delayed. The histone modification
Acode@ has not yet been demonstrated to be heritable. The true function
of histone modification patterns remains to be determined, and there was
disagreement as to whether an active role in determining chromatin
functions has been convincingly demonstrated, as opposed to a more
passive role as a result of replication or transcription.

The question of what epigenetic information is preserved through
replication, as well as the related question of what modifications are
causative of chromatin organization as opposed to simply the more
passive result of replication, were the topics of extensive discussion.
The roles of DNA methylation and histone modifications were discussed in
this light.

A critical next step is to determine the mechanisms of the replication
of specific chromatin structure, identifying the DNA sequences and the
targeting of chromatin remodeling factors involved in setting up
specific chromatin structures during or after DNA replication. Part of
the same effort will be to test the role of histone modifications in
epigenetic inheritance through the cell cycle, and to determine the
dynamics of histone modifications during DNA replication and the cell
cycle. We must deepen our understanding of how DNA methylation relates
to gene expression and chromatin structure.

*Epigenetic remodeling and genome stability*

It will be important to determine the mechanisms of regulating or
reversing the more stable epigenetic marks. For example, DNA methylation
can be either an active or a passive process, as discussed above, and
once methylated DNA demethylation may be via an active or passive
mechanism. Also, when histones are replaced with variants that have
different functions or modification sites, are these mechanisms active
regulatory mechanisms or secondary results of other functions? In any
case, it remains unknown if they have relevance to cancer.

Chromosomal rearrangements are a common feature of a wide variety of
neoplastic lesions and are thought to have a causal role in
tumorigenesis. Genome instability, leading to carcinogenesis, may occur
due to the genome-wide DNA hypomethylation observed in aging. Global
hypomethylation is frequently observed in tumor cells, and has been
associated with abnormal chromosomal structures, as observed in cells
from patients with ICF (Immunodeficiency, Centromeric instability and
Facial abnormalities) syndrome. However, the mechanistic link between
genomic instability and hypomethylated genomic DNA remains to be
discovered.

It will be very important to determine the mechanism for the creation of
neocentromeres. Centromeres are key players in genome integrity, and
defects in their function results in mis-segregation of chromosomes and
aneuploidy. Neocentromeres are ectopic centromeres that originate
occasionally despite the complete absence of normal centromeric
alpha-satellite DNA in the regions. No alteration of DNA sequence
occurs, and it remains unknown how they are created, but they are still
able to form a primary constriction and assemble a functional
kinetochore. Neocentromeres have been detected in certain cancers, and
are thought to be generally detrimental. However, no systematic
screening for these structures has been conducted.

Finally, it will be important to investigate the role of chromosome
territories, and factors that organize these territories, in
recombination and genome instability. Chromatin must be viewed in the
context of whole chromosome territories, organized into specific nuclear
zones. These territories occupy non-random positions in the interphase
nucleus. The mechanisms confining each chromosome in a defined territory
remains unknown, however, as well as the question of whether changes in
the position of the territories occur during differentiation or
carcinogenesis. The specific location of genes within nuclei is
important for proper gene expression, and nuclear architecture
contributes to the localization of genes. Thus, improper positioning of
genes may contribute to their improper regulation, potentially leading
to cancer. Cancer is associated with disruption of gene expression
patterns and global disorganization of chromosome organization within
the nucleus. The roles of nuclear architecture, including chromosome
territories, in genome stability and proper gene regulation must be
studied in depth.

**Epigenomics, Technology, and Translational Opportunities**

We are presently approaching the stage where we can begin to
"genomicize" epigenetic information. At the think tank, much of the
discussion of needed technology and translational opportunities took
place in the context of developing a Human Epigenome Project, analogous
to the Genome Project. The science is clearly developed to a point that
it is timely to plan a strategy for such an effort. The key question is
what information the efforts should focus on obtaining.

There are two decisions that need to be made before launching into a
large-scale effort. First, what epigenetic information should be
gathered and incorporated into the Epigenome? DNA methylation is the
first consideration, and most of the investigators want to make
genome-wide DNA methylation profiling using microarrays the top
priority. After that, other important elements that should and could be
incorporated include unequal allelic gene expression (including
imprinting); histone variants and modifications; chromatin organizer
proteins; and *cis*-acting marks for epigenetic signals. It must be
remembered that different types of epigenetic information present
different challenges. For example, the two fundamental epigenetic marks,
histone modifications and DNA methylation, differ in stability; certain
histone modifications can change quickly (acetylation reversed by
deacetylases, methylation reversed by histone replacement, etc.), while
CpG methylation of DNA is more stable.

Second, what tissue samples should be used? Unlike DNA sequence,
epigenetic marks and states certainly differ between tissues and change
with the age of the donor. A discussion is needed about what we know and
what we need to know, to permit these choices to be made. It would be
useful to ask what relationship exists between diversity in the
population and diversity in epigenetic marks, and what new epigenetic
population-based and family-based algorithms need to be developed.

*Key Issues and Potential Pitfalls*

New technologies are needed to study some questions of higher order
chromatin organization and function, and broader training is needed in
current state-of-the-art procedures such as the loop capture assay or
the ChIP-on-chip assay. Nonetheless, current methodology is sufficient
for conducting genome-wide screens of much of the epigenetic
information. Integration of epigenetic information into existing genome
databases would be optimal. A computer screen that incorporates all
levels of epigenetic information for each stretch of DNA sequence in the
genome was envisioned by some of the panel. In principle, data from
genome-wide or chromosome-wide bisulfite conversion/sequencing, histone
modifications, and mRNA expression profiling, can be overlaid on the
existing human genome sequence, to provide an integrated picture of the
genome and epigenome. This information, with associated annotations,
could be displayed at a resolution ranging from the whole chromosome to
the individual nucleotide in a clickable format, simply by adding a
series of new Ainformation fields@ to available, widely used genomic
databases. With this coordination of data, the project would begin to
provide answers about the potential for epigenetic therapy and how the
epigenome can be targeted for chemotherapy or chemoprevention.

Discussion of precisely what epigenetic marks and what tissues would be
included first did not produce a consensus at this meeting. All agreed
that future organizational meetings would be required to plan an
Epigenome Project. This project should take an approach somewhat like
the Human Genome Project, with a sequencing effort that provided
baseline information on which further genetic variation could be
identified. A comprehensive analysis of DNA methylation is needed,
including quantitative variation in the density and breadth (i.e.
numbers of modified residues) of methylation marks. In addition, novel
bioinformatic approaches must be developed.

In contrast to the DNA sequence, the epigenome varies among different
normal cell types and, perhaps more dramatically, between normal and
cancer cells. So, selection of the Areference cells@ and Areference
tissues@ for analysis in the Epigenome must be done with extreme care.
To deal with the problem of cell heterogeneity, microdissection will be
essential in preparing the samples. An even more fundamental issue
concerns the identification of the closest normal counterpart for any
give human cancer type, i.e. intelligent and meaningful selection of the
Acontrol@ samples for comparing to the cancer samples. Significant
progress is being made in this area, largely through gene expression
profiling and immunophenotyping, and this information needs to be taken
into account in making this critical decision on sample selection. It
would be a disastrous waste of resources to analyze the epigenome in
tissue samples that subsequently prove to be poorly defined and
heterogeneous.

*Feasibility of a Human Epigenome Project*

Despite these issues to be worked out, it was the consensus of the panel
that beginning an Epigenome Project is feasible now. Methods for
determining the pattern of CpG methylation of DNA and corresponding
patterns of histone modifications across the human genome are well
established. Chromatin immunoprecipitation/ChIP-on-chip assays can
rapidly score histone modification, potentially tiled across an entire
genome, and bisulfite conversion/DNA sequencing, as well as several
complementary approaches including microarray-based methods and genome
fractionation using McrBC endonuclease, can achieve the same objective
for DNA methylation. Furthermore, such a Project will be highly
leveraged by the completed Human Genome Project.

The most useful next steps towards beginning a Human Epigenome Project
were discussed. It will be crucial to identify epigenetic markers other
than methylation, and develop high-throughput tools to target and
characterize these markers fully. Also, it will be necessary to identify
new compounds and reagents, and compare these agents against
standardized reference sets. The tissue bank needed for the project must
be designed carefully. It must contain adequate amounts of material
across appropriate populations, and have samples varying in age, tissue
type, and disease state. A significant consideration is how banked
tissue samples should be processed and stored.

A hypothetical organization of a Human Epigenome Project would include
several Core Facilities, as well as a number of Pilot Projects that
would be expected to leverage the epigenetic data in specific research
areas:

[Core Facilities]{.underline}:

DNA Methylation Core

Histone Modification Core

Gene Expression Core

Bioinformatics Core (interfaced with the CaBIG consortium
[http://cabig.nci.nih.gov/caBIG/]{.underline} )

Tissue/Pathology Core

[Pilot Projects including but not restricted to]{.underline}:

Genome Imprinting and Allelic Asymmetry

Interindividual Variation in Epigenetic Marks

Mechanisms of Epigenetic Marking

Environmental Effects on the Epigenome

Cancer Detection using Epigenomic Targets

Epigenomic Drug Targets in Cancer Treatment

Bioinformatic Approaches to Epigenome Analysis

*Benefits of a Human Epigenome Project*

Epigenetic marks, defined as modifications of the genome that can be
faithfully propagated through cell divisions, and that affect central
biological processes like gene expression and genome stability, have
been amply shown to influence both cancer predisposition and tumor
progression. But in contrast to the DNA sequence, our understanding of
these epigenetic marks remains fragmentary, with most if not all of the
available data coming from highly selected regions of the genome,
examined by non-exhaustive methods. It is likely that a comprehensive
analysis of the epigenome, carried out in carefully chosen and well
defined normal and cancerous cells/tissues, will produce some truly
surprising and paradigm-shifting results, and may well yield fundamental
insights into cancer pathogenesis, detection and treatment. This
integrated epigenomic information will allow us to begin answering the
question of what is the basis for epigenetic therapy and how can the
epigenome be targeted for chemotherapy or chemoprevention.

Following some discussion of the European Epigenomic Network of
Excellence (see below), Think Tank participants discussed pursuing a
similar effort in the United States that would comprise a significant,
dedicated investment whose management and direction would be formulated
by an established network or committee. The justification for pursuing
such a project included:

\$ The series of discoveries in the last 10 years showing that
epigenetics is central to understanding of the development and genetics
of cancer.

\$ Moving forward with an Epigenome Project is timely in light of the
completion and findings of the Human Genome Project.

\$ To gain a better understanding of human disease, including cancer,
investigators need to take a genome-wide perspective of epigenetics.

\$ It will facilitate the standardization and dissemination of
information across the research community.

\$ It will increase visibility of the research community as a
coordinated group.

\$ Improved cost effectiveness, decreased redundancy, and better
comparisons are expected outcomes with standardization across research
teams, labs, and groups.

\$ Keeping the project in the public domain would help not only to
maintain but to encourage openness of communication, development, and
discoveries, including technological developments and advances.

\$ This strategy would provide an infrastructure for reviewers to
improve genome study sections through increased awareness, expertise,
and innovation.

\$ It will provide a much-needed, coordinated opportunity for
quantitative/statistical epigenetics and the development of new tests,
tools, and measures.

\$ It will provide a missing component of integrative biology.

> **Special Presentation: European Union 6^th^ Framework ProgrammeCThe
> Epigenome**
>
> Dr. Patrick Varga-Weisz summarized the EU=s Epigenomic Research
> Program, which provides a platform and structure to facilitate the
> coordination of epigenetics research beyond local efforts. The program
> takes advantage of established research groups and facilitates the
> development of new research talent. A Acore consortium@ of 25 leading
> scientists in epigenetic research receives funding through a Joint
> Program of Activities (JPA) for the first phase of the Epigenomic
> Network of Excellence (NoE), which will run from 2004 through 2009
> with an annual budget of approximately 12.5 million Euros.
>
> The purpose of the Epigenome Program is to integrate European
> epigenetics and epigenome research; to support new researchers; and to
> facilitate dialogue among investigators. More specifically, the three
> key goals are to coordinate and support:
>
> ● European epigenetic research: To unravel epigenetic mechanisms in
> the post-genomic era, the NoE will prioritize research into molecular
> mechanisms of epigenetic control. To achieve this goal, the NoE will
> mount an internationally competitive research program through a Joint
> Program of Activities (JPA).
>
> ● The next generation of European scientists: A funding program for 22
> independent investigators will distribute monies in two rounds over 5
> years to integrate the most promising newly established teams (NET
> program) in Europe.
>
> ● An interactive Website: Scientists and the public will be provided
> with up-to-date knowledge of the field through a Web site maintained
> by the NoE. This Web site \[for the preliminary Web site, go to
> http://www.epigenome.imp.ac.at/index.html\] will develop into a major
> resource for the scientific community, and it will seek to establish a
> dialogue with the public by providing first hand knowledge in an
> appropriate form for a non-specialist audience.
>
> The NOE research program is organized into eight distinct subprograms
> focusing on different aspects of epigenetic control:
>
> ● Chromatin modification
>
> ● Nucleosome dynamics
>
> ● Non-coding RNA and gene silencing
>
> ● Xi and imprinting
>
> ● Transcriptional memory
>
> ● Assembly and nuclear organization
>
> ● Cell fate and disease
>
> ● Epigenomic maps

**Specific Recommendations for the NCI:**

*Specific major recommendations*

1\. Establish a Human Epigenome Project of analogous scope to the Genome
Project. This was the highest priority recommendation. The Project could
include:

> \$ Developing a baseline of epigenetic information and profiles that
> includes reference samples, and expanding exploration of integrative,
> comprehensive epigenetic profiles of normal and tumor tissues.
>
> \$ Developing detection tools to genomicize epigenetics; i.e., to lead
> to robust, cost-effective, high-throughput, genome-level analysis.
>
> \$ Developing novel bioinformatic approaches to epigenetics, including
> computational and statistical tools and comparative epigenomics. A
> computer screen which incorporates all levels of genetic/epigenetic
> and chromatin knowledge was envisioned.

2\. Organize a planning meeting, and establish a Working Group, to
define the needs for a Human Epigenome Project. This was unanimously
agreed upon. The questions to be determined include: tissues to be used;
how many individuals; relationship to Genome Map; what will be measured
B DNA methylation, histone modifications, other chromatin proteins

*General recommendations*

3\. Support technology development for epigenetic profiling as well as
for studies of basic chromatin organization is needed. This may be
addressed as part of Epigenome Project; or may relate to Recommendation
4 below; or may require an R21 initiative to develop technology.

4\. Encourage and foster R01-based epigenetic research, particularly for
basic research in mechanisms of chromatin structure and gene regulation.

5\. Encourage and support translational research with a focus on early
epigenetic variations and translational consequences, e.g., how do these
variations alter adult susceptibility to disease?

6\. Expand training opportunities, including cross-platform training for
post-docs, junior and senior faculty, and institute U.S.-European
collaborations or cross-training agreements.

**Executive Summary of Inflammation and Cancer Think Tank**

 

Inflammation is a response to acute tissue damage, whether resulting
from physical injury, ischemic injury, infection, exposure to toxins, or
other types of trauma. It can play a role in tumor suppression by
stimulating an antitumor immune response, but more often it appears to
stimulate tumor development. Epidemiologic and clinical research
indicates an increased risk of certain cancers in the setting of chronic
inflammation.  For example, two inflammatory bowel diseases, ulcerative
colitis and Crohn's disease, predispose to cancers of the intestinal
tract.  Basic research, in turn, has shown that many of the processes
involved in inflammation (e.g., leukocyte migration, dilatation of local
vasculature with increased permeability and blood flow, angiogenesis),
when found in association with tumors, are more likely to contribute to
tumor growth, progression, and metastasis than to elicit an effective
host anti-tumor response.

Interestingly, inflammation functions at all three stages of tumor
development: initiation, progression and metastasis. Inflammation
contributes to initiation by inducing the release of a variety of
cytokines and chemokines that alert the vasculature to release
inflammatory cells and factors into the tissue milieu, thereby causing
oxidative damage, DNA mutations, and other changes in the
microenvironment, making it more conducive to cell transformation,
increased survival and proliferation.

Chronic inflammation appears to contribute to tumor progression by
establishing a milieu conducive to development of different cancers.
However the precise mechanism by which it does so remains to be
determined. Infection is a common cause of inflammation, and evidence
indicates that the presence of microbes can be a cofactor in the tumor
promoting effects of inflammation.

Tumor cells produce various substances that attract inflammatory cells,
which then secrete an array of soluble mediators. These further
stimulate proliferation of the initiated cell, tissue disruption in the
stroma, and tumor growth. Leukocyte infiltration, and particularly
macrophages, can lead to enhanced angiogenesis, which is associated with
a poor prognosis in some tissues. 

The role of inflammation in metastasis is less well defined than its
roles in cancer initiation and progression. The soluble mediators
secreted by tumor-associated leukocytes promote cell motility, and
induce angiogenesis, vascular dilation and extravasation of tumor cells.
Particularly interesting is the recent finding that metastatic cells
leave the tumor as microcolonies, containing lymphocytes and platelets
as well as the tumor cell. Inflammation continues to play a role at
metastatic sites by creating a cytokine milieu conducive to tumor
growth.

Although there is a strong association between chronic inflammation and
cancer, investigators have not yet uncovered all the molecules,
pathways, and mechanisms involved, and numerous questions remain to be
resolved about the mechanisms and targets of pro-inflammatory mediators
of tumor development. These are articulated in the body of the report
and the recommendations that follow. Furthermore, to understand the role
of inflammation in tumor formation and progression, we also need to
understand its role in maintaining homeostasis and responding to damage
in normal tissue. An appreciation of the importance of inflammation has
already led to clinical trials of anti-inflammatory drugs (e.g., COX-2
inhibitors) for cancer prophylaxis and treatment. The results obtained
will provide clues to the dominant mechanisms at work, and will help in
the design of a new generation of interventions.

**Introduction**

Inflammation involves a complex set of interactions between soluble
mediators and immunocytes, triggered in response to tissue injuries that
include trauma, infection, toxic agents and autoimmune responses. Such
injuries trigger a cascade of cellular infiltrations and cytokine
releases that result in local cellular proliferation and repair of
tissue damaged. While sustained proliferation alone is insufficient to
initiate cancer, a functional relationship between inflammation and
cancer has been recognized for a long time. The current discussions
centered on our current understanding of the role of inflammation in
cancer initiation, progression and metastasis and highlighted areas in
which there are major, unresolved questions.

# Discussion Themes

***I. Inflammation and Cancer Initiation***

Although inflammation is a necessary response to clear viral infections,
to repair tissue insults - either chemical exposure or injury- and
suppress tumor initiation/progression, chronic inflammation is also
clearly correlated with increased risk of developing cancer.
Inflammation may become chronic either because an inflammatory stimulus
persists or because of dysregulation in the control mechanisms that
normally turn the process off. Many of the cells, cytokines and systems
(e.g., leukocyte migration, dilatation of the local vasculature and
angiogenesis) involved in inflammation are also found in a variety of
tumors. Chronic inflammation caused by intestinal flora leading to the
inflammatory bowel diseases, ulcerative colitis and Crohn's disease, is
clearly linked with a higher incidence of colon cancer. The use of mouse
models has furthered our understanding of the contributing cellular and
molecular factors in colon cancer. Similarly, dietary intake of
proinflammatory carcinogens has been associated with prostate cancer.
Chronic inflammation resulting from esophageal reflux gives rise to
gastroesophageal reflux disease (GERD) and Barrett's esophagus, also
linked with a higher incidence of cancer. In the case of Barrett's,
chronic inflammation leads to the production of TNF. This, in turn,
induces the nuclear translocation of -catenin and transcriptional
activation of proliferative signals.

The molecular basis for the increased risk is thought to be two-fold: 1)
generation by inflammatory macrophages of reactive oxygen (ROS) and
nitrogen (RNS) species leads to DNA damage in the surrounding epithelial
cells and 2) enhanced proliferative signals mediated by cytokines
released by inflammatory cells increase the number of cells at risk for
mutations. In combination, DNA damage and proliferative signals create a
circumstance conducive to the development of cancer. ROS and RNS can
cause extensive damage to essential proteins (e.g., DNA repair enzymes),
to DNA and to the mitochondria through a series or cascade of reactions.
Among the many possible mutations that may result from oxidative DNA
damage are the formation of single- and/or double-stranded breaks and
the stimulation of recombination events. Free-radical damage can be
caused by the pro-inflammatory prostaglandin enzyme, cyclooxygenase 2
(COX-2), which leads to the production of highly reactive peroxide
intermediates at high levels in a local tissue environment. Drugs that
selectively inhibit the COX-2 enzyme, including NSAIDs, are being
studied to determine their impact on local tumor biology and
development, and in clinical trials. Recent studies have suggested
protective effects of COX-2 inhibitors in colorectal cancer and breast
cancer. Several small studies of colorectal, non-small cell lung cancer,
breast, cervical and esophageal tumors have shown that increased COX-2
levels are associated with poor clinical prognosis. Animal models for
colorectal cancer show similar patterns of COX-2 expression and response
to COX-2 inhibitors as human neoplasias.

Inflammation results in the recruitment of leukocytes secreting a
variety of proliferative cytokines and angiogenic factors to the site of
tissue insult. These cytokines, necessary for proper wound healing,
stimulate epithelial proliferation, which if unchecked could lead to
dysplasias and ultimately cancer. Paradoxically, cytokine deficiency
(e.g., GM-CSF, IL-2 and IFN) can also lead to tumor development. Immune
homeostasis consists of a succession of pro- and anti-inflammatory
signals. Loss of the anti-inflammatory signals leads to chronic
inflammation and proliferative signaling. The mechanisms involved in the
interplay of microbes and defective immune homeostasis is an area that
requires further delineation. Future steps will involve clinical studies
to determine whether individuals have polymorphisms or genetic
variations that affect specific cytokine pathways.

The discussion highlighted the duality of inflammation in controlling
and promoting tumor development. While chronic inflammation can
establish conditions conducive to tumor initiation and progression,
compelling data also suggest that the presence of lymphocytic
infiltrates in a variety of tumors is associated with a good clinical
outcome. The major challenge in this area is to understand the balance
between inflammatory tumor suppression and promotion and how to control
it.

***II. Inflammation and Cancer Progression***

It is generally accepted that chronic inflammation -- triggered by
toxins, microbes or autoimmune reactions -- plays a major role as a
tumor promoter. However, the precise function of inflammation in tumor
progression remains to be elucidated. Tumor cells produce various
cytokines and chemokines that attract leukocytes, which in turn produce
cytokines and chemokines that stimulate further tumor cell
proliferation; the inflammatory tumor microenvironment is characterized
by the presence of host leukocytes both in the stroma and around the
tumor. A developing neoplasm can contain a diverse leukocyte population,
including neutrophils, dendritic cells, macrophages, eosinophils, mast
cells and lymphocytes. These inflammatory cells secrete an array of
cytokines, interleukins, interferons and other soluble mediators and
further induce secretion of cytokines by resident stromal cells.

Interestingly, both cytokines that promote and suppress proliferation of
the tumor cells are produced. As in the case of cancer initiation, it is
the imbalance between the effects of these two classes of activity that
results in tumor promotion. For example, in the presence of GM-CSF and
IL-4, monocytes differentiate into immature dendritic cells, which
migrate into inflamed peripheral tissue, capture antigens and then
migrate to lymph nodes to stimulate T lymphocyte activation. Deletion of
GM-CSF from Polyoma T transgenic mice reduces cancer progression. This
is correlated with reduced macrophage infiltrates, which play a major
role in the transition from adenoma to carcinoma. GM-CSF vaccines that
stimulate dendritic cell and T cell responses are being combined with
anti-CTLA4 treatment in clinical trials to potentiate the anti-tumor
response.

In contrast, IL-6 and CSF-1 secreted by tumor cells can skew monocyte
differentiation towards the macrophage lineage. Although tumor
associated macrophages can kill tumor cells when activated by IL-2,
IL-12 or interferon, they also produce a host of compounds -- angiogenic
factors, growth factors, proteases and cytokines -- that either
contribute to cancer progression or blunt the anti-tumor response.
Macrophages generate a variety of proteases, including cathepsin B,
which contribute to tumor growth. Stromal fibroblasts and monocytes
enhance this proteolysis. During tumor progression, the degradation of
the matrix and stromal fibroblasts appears to be focal, suggesting that
widespread degradation may not be necessary for tumor growth. In mammary
cancer models, a variety of leukocytes are found at the tumor-stroma
interface. A complex interaction between tumor, stroma and inflammatory
cells results in the secretion of protease and matrix degradation and
the entrapment and degradation of fibroblasts by the tumor.

Other studies have indicated that hypoxia signaling pathways are engaged
very early in cancer development. Hypoxia stabilizes HIF-1 which in
turn induces VEGF secretion by epithelial cells that stimulates
microvascularization and angiogenesis.

The spatial relationship between inflammatory cells and tumors is now
being investigated by imaging of a range of live human tumor and
associated cells. Examination of the interaction reveals that although T
cells will home to the tumor, they stay on the periphery and do not
enter. Real-time images of mammary tumors in mice show that the tumor
regions are metabolically active, compared with surrounding stroma and
fat cells. These regions also have a much greater inflammatory response;
associated immune cells, particulary T cells, are very active and
mobile. In mammary cancer models, imaging reveals that tumor associated
macrophages preferentially line up along the lumenal side of the
tumor-associated vessels. Macrophages in this region are relatively
static, whereas those at the stromal interface are very active and
motile, suggesting differential behavior within the tumor. The
functional significance of this remains to be determined.

Although the role of inflammatory cells and soluble mediators in tumor
progression is now well documented, the details of the cellular and
molecular interplay between stroma and tumor progression remain to be
elucidated.

***III. Inflammation and Metastasis***

Unlike tumor progression, where the role of inflammation in promoting
cancer cell proliferation and stromal/matrix degradation is reasonably
well understood, the role of inflammation in metastasis is less well
defined, although appears to be important. The cytokines and chemokines
secreted by tumor associated macrophages and leukocytes promote cell
motility and induce angiogenesis and the growth of tumor-associated
vessels, providing an egress route for metastatic tumor cells. The
leukocytes also promote vessel dilation and extravasation of tumor
cells. Particularly intriguing is the observation that metastatic cells
leave the tumor as microcolonies, containing lymphocytes and platelets,
the latter allowing attachment to distal organ sites. Tumors that are
unable to form such microcolonies are not malignant.

At the distal, metastatic sites, evidence suggests that inflammation
continues to play a role in the establishment of metastases. At the
sites of prostate cancer metastases in the bone, inflammation triggers
the secretion of TGF-β by osteoclasts. TGF-β in turn induces the cancer
cells to secrete PDGF which further stimulates the osteoclasts, leading
to bone degradation and stimulation of cancer cell growth. PDGFR on
tumor-associated endothelial cells increases their levels of bcl-2 and
bcl-xl, rendering them resistant to apoptosis and chemotherapy.
Similarly, in brain metastases of melanoma, astrocytic infiltrates
upregulate MDR (multiple drug resistance) in the tumor cells, making
them more resistant to chemotherapy.

**Next Steps and Important Questions**

-   Despite the evidence for the role of inflammation in cancer
    intiation, progression and metastases, other evidence suggests that
    cancer proceeds through inflammation-dependent and independent
    stages. These stages need to be defined and characterized.

-   To better understand the critical role of inflammation in initiating
    and modulating tumor behavior, host-pathogen interactions need to be
    defined at a molecular level, the phenotypes of hematopoeitic cells
    (leukocytes, monocytes, platelets, etc) involved in wound repair and
    tumor initiation need to be characterized and the roles of
    endocrinological mediators on inflammation need to be examined.

-   To better understand the role of inflammation in tumor progression,
    tumor stage should be correlated with intensity and repertoire of
    hematopoietic infiltrates and with the levels of cytokines and
    proteases present; biomarkers for pre-malignant and malignant
    lesions need to be identified and validated.

-   To better understand the role of inflammation in metastasis, better
    pre-clinical models need to be developed, the molecular relationship
    between primary and metastatic tumor cells needs to be resolved, the
    nature of the inflammatory responses that influence primary versus
    metastatic tumor need to be determined, and the role of the
    hematopoietic network in tumor extravasation and migration needs to
    be elucidated.

-   Real-time imaging models need to be refined and extended to allow
    better definition of the relationship between inflammatory and tumor
    cells that influence the cancer initiation, progression and
    metastasis process.

> Tumor immunotherapy approaches should include targeted intervention of
> inflammation-mediated growth, pairing molecular information of
> inflammatory infiltrates with that of specific tumors. Therapy should
> be specifically directed to both the organ microenvironment and the
> tumor.
>
> **Specific Recommendations for the NCI:**

-   Continue to support basic research aimed at characterizing the role
    of inflammation at all stages of cancer and specifically at
    determining the role of hematopoeitic cells in both metastasis and
    normal development.

-   Encourage collaborative studies between basic and clinical
    investigators. Support studies that characterize inflammatory cells
    in the tumor microenvironment and correlate these with clinical
    outcomes and prognosis

-   Consider mechanisms to stimulate multi-agency, multi-institutional
    and transdisciplinary collaborations to more rigorously define
    critical interactions that occur between tumor cells and their
    inflammatory microenvironment.

-   Sponsor a series of workshops and/or interactive fora on
    inflammation and cancer that interface experts from different
    disciplines (i.e., toxicologists, cellular and tumor immunologists,
    systems biologists, cancer biologists).

-   Develop and standardize reagents and protocols for analysis of
    archived tissues. Establish a uniform database of existing reagents
    and well-defined and catalogued tumor types that are or are not
    associated with inflammation.

-   Establish and make available conditional tissue- or cell-specific
    pre-clinical models to study the biology of inflammation-dependent
    cancers and to stimulate novel prevention, diagnostic and
    therapeutic strategies.

-   Consider mechanisms to provide investigators with

    -   access to imaging tools for in vitro and in vivo analysis to
        characterize immunocyte/tumor interactions.

    -   more sophisticated and cheaper imaging modalities, perhaps by
        encouraging development in the private sector through the SBIR
        mechanism

    -   training in the use and application of imaging techniques

**Executive Summary of the NCI Think Tank on Variation in Susceptibility
to Cancers**

At present, we assume that cancer develops in a particular person
because of her/his unique genetic composition and exposures, and the
complex interplay among them. Intensive studies of strongly cancer-prone
families inheriting the same mutant allele of a cancer gene illustrate
that more than just pre-disposing genes confer and modify risk of
developing cancer. Many parameters of the malignancy -- severity,
timing, therapeutic response, and even manifestation of the cancer
itself -- may differ markedly among cancer-prone siblings and between
generations. Technological advances enable us to identify many of the
familial and non-familial genes and aberrant processes that confer some
of the susceptibility to cancer, and to assess the probability that
certain environmental exposures contribute to the likelihood of disease.
However, we require a fuller exposition of the genetic architecture of
cancers as complex traits if we are to define population and individual
cancer risk more accurately. Genetic architecture is the fusion of the
effects of interactions among genes and environmental perturbants; it is
thus more than the sum of those individual factors. To achieve
significant advances in insight about cancer risk, we must propose and
test new theoretical models of genetic architecture and use experimental
model systems to test the resulting hypotheses about individual
susceptibility and resistance factors, their interactions, and their
manifestations. This will require that we redirect many of our on-going
epidemiologic and cancer modeling efforts toward coordinate activities
that play to the strengths of each research community and its unique
contribution to the collective effort.

In addition to epidemiologists and cancer modelers, the scientific
redirection includes augmenting the intellectual inputs to include
computational biologists, mathematical modelers, and evolutionary
biologists, among others. To encourage and sustain the new interactions
requires access to human specimens and data, development of new
analytical strategies to tease apart the contributors to genetic
architecture and new algorithms to model genetic and non-genetic
interactions, interdisciplinary training in computational analysis and
modeling, and computer systems capable of rapid high-level data
analyses.

**Vision** and **Overall Goal:** To understand the complex interactions
of genetic and environmental influences; to predict the risks of cancers
for individuals and populations; to enhance prevention, perfect
diagnosis, target treatment, improve prognosis prediction, and greatly
reduce the burden of cancers.

To achieve these goals, the scientific community must:

-   Integrate studies of experimental model systems, human populations,
    and computational models of molecular signatures in normal and
    dysregulated states relevant to particular features of cancer
    susceptibility.

-   Improve cancer-related phenotyping (i.e., the analysis of molecular
    and systemic changes in cancer progression) by applying emerging
    sensor, imaging, nano-and molecular technologies to individual
    humans and animals, biological specimens, cells, extracellular
    matrix components, and cell-cell interactions.

-   Identify sources of individual variation in cancer-related
    biological processes, both genetic and non-genetic, and model the
    genetic-environmental and stochastic interactions.

-   Develop and apply technologies to investigate genetic variation
    broadly, including alleles, SNPs, haplotypes, and epigenetic
    alterations of nuclear and mitochondrial genomes, miRNAs, highly
    repetitive sequences, transposons, gene copy number polymorphisms
    and other evolutionary molecular features.

**Roadblocks and Challenges** that currently hamper progress in
understanding cancer susceptibility were identified. ***Roadblocks
include*** the cost and limited scope of current high-throughput
technologies for the molecular characterization of tissues and tumors
and the genotyping of individuals; the inadequate scope of systemic
clinical measures of cancer initiation and progression; the difficulty
of assessing environmental exposures relevant to cancer development; the
lack of an adequately large human cohort for prospective studies of
susceptibility, along with the necessary infrastructure and biological
repositories; the need for long-standing informed consent valid for
multiple studies; and the serious lack of infrastructure and
requirements for data sharing and standardization. ***The major
challenges are*** the development of new statistical and computational
approaches adequate to deal with the level of complexity believed to
exist in cancer susceptibility, involving multiple genes and
environmental factors, interacting in complex, non-linear ways; the
integration of human studies with those in experimental systems to
optimize understanding of carcinogenic mechanisms; and developing
interdisciplinary training and research environments that incorporate
the perspectives of biologists and computational scientists.

**Introduction**

The overall goal of defining cancer susceptibility in terms of the
interaction of genetic variation and environmental factors is an
ambitious one that cannot be achieved with a single line of
investigation. It will require integrating studies in experimental model
systems, human populations, and computational models. Studies are
required not only on frank malignancies, but on normal physiology and
dysregulated states along the continuum from the earliest phase of
initiation through metastasis. Several specific requirements were
identified:

**1. Improve cancer-related phenotyping (i.e., the analysis of molecular
and systemic changes in cancer progression) by applying emerging sensor,
imaging, nano-and molecular technologies to individual humans and
animals, biological specimens, cells, extracellular matrix components,
and cell-cell interactions.**

Cancers are a very heterogeneous collection of diseases; correlating
phenotype with genotype depends on a detailed knowledge of the clinical
manifestations of each cancer type. Susceptibility studies are designed
to find and test associations between genotype and a specific cancer
phenotype. Early studies of tumor classification through the use of DNA
microarrays, as in NCI's Director's Challenge program, suggest that
molecular phenotyping is a powerful approach to stratify cancers in ways
not possible using histopathology and the few previously available
molecular markers of cancer behavior. Such disease stratification will
sharpen the ability to study susceptibility.

Although microarray studies are useful, and allow researchers to
ascertain the expression level of thousands of genes simultaneously,
expression analysis alone does not meet the need for a complete
phenotyping program. Techniques to expose epigenetic changes and to
define the serum and cellular proteome are the next frontiers. Studies
of epigenetic change are important because DNA sequence is not the only
heritable change in cells and individuals. Proteomics is important
because mRNA levels correlate imperfectly with protein levels and
proteins are subject to many post-translational modifications that
strongly influence their functions. Metabolomics will become
increasingly important as the field of cancer phenotyping matures.
Moreover, there is a need to evaluate not only absolute levels of genes
and gene products, but their rates of change.

Studies of normal tissues and the tumor microenvironment further
demonstrate that it is insufficient to determine the phenotype of the
cancer cell; the properties of tissues and alterations of those
properties as tumors emerge are what is most relevant. The behavior of a
cancer cell is influenced by the cells and molecules with which it has
contact; fibroblasts, infiltrating lymphoid and myeloid cells,
endothelial cells, the extracellular matrix, and soluble molecules, such
as growth factors, cytokines, chemokines, nutrients, and oxygen, all
influence tumor phenotypes.

Characterizing tumor phenotypes at this level will benefit many areas of
cancer research, and such studies are proceeding. However, it is
worthwhile emphasizing how critical these studies are for susceptibility
research.

**2. Examine all cancer-related biological processes for sources of
individual variation, both genetic and non-genetic, and model the
genetic-environmental and stochastic interactions of genetic networks
and cancer phenotypes.**

Susceptibility to cancer results from both genetic and non-genetic
individual variation within a human population. Genetic variation is
commonly equated with polymorphisms in germline DNA sequence, but this
is an over-simplification. Among other potentially relevant genetic
complexities are: Imprinting; X-chromosome inactivation; mitochondrial
DNA; segmental inversions; deletion or alteration of regulatory
elements; jumping elements; tri-nucleotide repeats; and others. Although
susceptibility to many cancers is influenced by multiple genes, a recent
study suggests that genetic variation is a minor component in cancer
susceptibility, contributing perhaps 25% to the risk. The rest is
attributable to a wide range of environmental factors, including
smoking, diet, hormonal status, chemical exposures, microbial flora, and
history of inflammation.

Environmental factors add unsolved methodological complexities to the
evaluation of cancer susceptibility. Genotypes can be determined with
high precision, but current ways of quantitating environmental exposures
generally do not capture all the information needed for a large study.
Dietary histories, for example, are notoriously inaccurate. Biomarkers
of most chemical exposures are lacking. Microbial flora and inflammation
are increasingly recognized as factors in cancer development, but no
consensus exists on which factors to measure or how to evaluate their
impact.

With so many challenging variables to deal with in susceptibility
studies, it is tempting to make the simplifying assumption that the
contribution of each susceptibility factor is independent. However, the
available evidence indicates that this would be a mistake. In *Epistasis
and the Evolutionary Process* (2000), Templeton states that, using
proper computational methods and algorithms, one finds complex
interactions (epistasis) more often than additive, single-factor
effects. In studying susceptibility, it is necessary to ask complex
questions and develop complex models. A shift away from single gene or
single environmental factor analysis to multi-gene, multi-factor
analysis is required, but current analytical and statistical tools are
inadequate to support such a shift.

**3. Develop and apply technologies to investigate genetic variation
broadly, including alleles, SNPs, haplotypes, epigenetic alterations of
nuclear and mitochondrial genomes, miRNA, and other factors as they are
identified.**

A few relatively high-throughput technologies exist to evaluate some
types of genetic variation, but improvements in existing technologies
and the development of new ones are required. Genome-scale sequencing
efforts mean that complete genome sequences for quite a number of people
will soon be available. One spin-off of this sequencing work has been
the development of methods for whole-genome scanning using
microsatellites, and now single-nucleotide polymorphisms (SNPs). SNP
typing is rapidly becoming the technology of choice for genotyping as
the cost is reduced, and the value of the technique will rise rapidly
with further cost reductions and the development of analysis techniques
that can handle the masses of data generated. The HapMap project leads
the way in identifying and validating human SNPs, and in organizing them
into haplotypes to maximize the information content of SNP mapping.
Similar efforts are underway for model organisms.

However, more progress is needed in other areas of genetic analysis.
High-throughput assessment of epigenetic variation is at least a decade
behind that of DNA sequence variation, and new techniques are required
to interrogate the genomic architecture for regulatory features. With
the discovery of potential new sources of genetic variation, such as
regulatory micro RNAs, the need for technology development continues to
increase.

**Specific Recommendations:**

**1. Given the scale of data sets and complex biological systems, create
new computational, mathematical, and statistical models and analytical
tools that** **embrace, rather than simplify or ignore, the complexity
of variation in** **susceptibility and resistance to cancers. Support
large-scale coordination of studies with calibration, interoperability,
data federation, and standardization of data formats, natural language
processing tools, and dataset query tools.**

Current analytical tools and approaches are inadequate to deal with the
complexities inherent in assessing susceptibility (see Requirement 2,
above). Orders of magnitude more data and types of data will need to be
acquired, assembled, and analyzed into information, and knowledge
extracted. This will require development of new approaches to
statistical analysis, integration of modeling efforts into the field,
and changes in how data are collected, annotated, and stored.

***Analytical Approaches-*** The currently accepted picture of cancer
susceptibility, involving multiple genetic and environmental factors
acting together in a non-additive fashion, cannot be adequately tested
experimentally because of limitations in statistical methods and
computational resources. These limitations necessitate the introduction
of simplifying assumptions that are likely to be invalid. As a result,
potentially critical variables are ignored or oversimplified. The types
of analytical methods that need to be developed involve combinatorial
problems. Epidemiological analysis is generally restricted to use of a
linear modeling paradigm, but such statistical methods are unsuitable
for the amount and complexity of data that will be needed to test
realistic hypotheses. It is necessary to shift from parametric methods
to those that deal more successfully with complex interactions.
Significant computational resources are required to deal with
high-dimensional data sets; although computing power is increasing, new
algorithms and strategies are needed to solve the problems and model
complex systems. The methods depend on non-linearity and non-additivity
and involve applied biostatistics, a field that moves forward slowly. To
anticipate the requirements of research five to ten years in the future,
research is needed now in the way statistical methods and design
algorithms are applied.

***Modeling-*** While simple problems can be analyzed directly, problems
as complex as cancer susceptibility must be computationally modeled to
understand what data needs to be collected and what analytical
approaches are likely to be successful. Modeling a biological process at
early stages of a project can provide information about the amount of
data needed to represent the process accurately, and ensure that enough
data is collected to make analyses feasible. Modeling can also help to
determine what types of interactions among variables are most likely to
affect the outcome, and focus computing power on the most productive
problems.

Models and experiments are refined in an iterative process that requires
regular interaction among model developers, model simulators, and
biologists. The proper integration of modeling into laboratory studies
or epidemiologic projects requires building a mutually beneficial
working relationship with experts in the quantitative discipline in
which modelers are typically trained. Because the empirical approach
relies heavily on statistics, empirical scientists and statisticians
have learned to communicate well. Similarly productive communication
between biologists and computational scientists is less common, limiting
the development of modeling.

***Data Issues: Standardization, Calibration, Test Validation, and
Integration***

Standardized nomenclature, controlled vocabularies, and natural language
processing are needed. Many data sets cannot be pooled because they are
not standardized. Data collection must be standardized -- especially in
the large-scale cohort (see Recommendation 3, below) -- and data sets
integrated so that comparisons can be made. One approach to
standardization and facile data sharing is to aggregate the necessary
technologies in one place. This might involve a consortium of many
academic institutions using the same standards, so that collection could
be correlated with sample analysis. Even modeling should be standardized
to enable investigators access to several well-defined pathways to work
with.

Calibration is critical for pooling of some data types, but it is
currently difficult because groups do not use the same controls, even
where controls are possible. In addition, in some fields such as
structural and functional MRI, no calibration exists to permit
cross-comparisons among instruments, so no valid comparisons are
possible.

Test validation is needed, especially for the most fundamental data
types. If thousands of SNPs will be typed on DNA samples, the procedure
must be validated. The Think Tank participants recommend obtaining a
uniform set of data using lymphoblastoid cells from a control population
for this process.

Finally, mechanisms are needed to integrate new data into what already
exists. Scientific communities need to pool information in ways that
maximize the use of the resources. If a large-scale cohort were to be
established, it will pose particular challenges of data integration
because the database is likely to be a federated system, assembled by
linking datasets at many separate locations. The community's computer
development effort needs to build integrated models that can maintain
security.

**2. Create a common and widely available repository with existing
resources of well-selected, maintained, and annotated human biological
specimens (immortalized lymphoblastoid cell lines, serum, plasma, urine,
etc.) for genetic, epigenetic, proteomic, and metabolomic analyses of
early-onset cancer cases, including those with positive family
histories. Create common databases. Develop a corresponding resource
from a special elderly subpopulation to study resistance to cancer
development in individuals with high lifetime exposure to carcinogens.
Require data deposition in freely accessible databases, with resource
sharing of repository materials.**

Developments in analytical techniques, modeling, and data collection and
storage, all described above, will become especially useful when applied
to large, highly informative population studies. The most ambitious such
proposal is described in the next section, but there was general
agreement at the Think Tank that at least two studies hold special
promise in the short term. The first is a study of early onset cancer
patients and their families, to look for particularly influential
susceptibility genes.

The second study would focus on cancer resistance, the opposite side of
susceptibility. The carcinogenic potential of smoking is very high, but
some heavy smokers live to be 100 years of age, and eighty-five percent
of smokers do *not* develop lung cancer. Family studies should be
developed based on unusual resistance to cancer. For example, Peter
Shields and others have cohorts of 90-year old smokers from Veterans'
Hospitals; these cohorts would be a valuable addition to a large cohort
study. Some individuals develop multiple cancers, but live a long life
because their tumors do not metastasize. The interactive genes that
confer apparent resistance to metastasis should be identified.
Susceptibility or resistance to progression or metastasis should receive
the same attention as susceptibility or resistance to initiation.

These two studies are important on their scientific merits, but they
also provide an opportunity to test developments that need to be applied
to all studies in cancer susceptibility. In addition, they can serve to
pilot high-quality repositories for biological samples, strategies for
dealing with privacy issues, and ways to mandate data and resource
sharing.

***Biological Resources/Repositories***

For these studies and the large cohort study described below, a
repository for human specimens is a necessity. A common resource of
biological specimens should be developed. This should include a standard
set for iterative studies, with enough samples for test training sets of
data. Repositories containing material useful in the study of cancer
susceptibility exist throughout the world, but their optimal use by the
scientific and clinical communities is hampered by impediments. Beyond
the existing repositories of samples from "common" types of cancer and
populations, susceptibility studies require cell lines from families to
investigate familial gene variation. The success of ongoing efforts by
the NCI to optimize the large-scale and efficient acquisition, storage,
annotation, and distribution of biological samples is critical to
progress in cancer susceptibility.

***Resource Sharing***

It is important to capitalize on existing resources, including large
datasets and large specimen stores, many of which have not yet been
analyzed. Investigators could contribute these samples for analysis, and
retain access to the data for a reasonable period of time (e.g., six
months) before the data enters the public domain. NCI could facilitate
such resource sharing by paying for processing the material.

***Privacy, Sample identification, and Patient Concerns***

New mandates for patient privacy provide challenges for the study of
cancer susceptibility. It is critical to retain some identification with
genetic information and to keep family data together, yet current
requirements to de-identify data impede progress in assessing
gene-environmental interactions. Informed consent is also a potential
problem. Durable informed consent must be obtained so that samples are
approved for varied, long-term uses. These issues generated considerable
discussion at the Think Tank, but the Think Tank participants were
optimistic that, given sufficient attention, these problems could be
dealt with.

***Data Sharing***

Little, if any, epidemiological data relevant to cancer susceptibility
is freely available for computer downloading. In addition, there are
hundreds of linkage studies in human genetics from which conclusions
have been published, but the underlying raw data remain unavailable. As
a result, experimentalists have no way to compare their data to that of
other labs, and computer scientists developing algorithms useful for
cancer research have no way to test them, short of entering into formal
collaborations. The NIH or NCI could, and should, exert pressure for
release into the public domain of raw data underlying published
analyses. In a number of large-scale research projects funded by NCI or
elsewhere at NIH, data release is required, and sometimes even
facilitated. This practice needs to be much more widespread, and
reasonable schedules for data release need to be negotiated with input
from the scientific community. In coordination with funding agencies and
professional societies, journals can facilitate data release by
requiring release of underlying data as a condition of publishing the
conclusions.

Datasets are unavailable because they are difficult and expensive to
produce. They can be reused by the original investigator in new studies,
and no incentives exist for sharing them. No credit is given in
university promotion and tenure decisions for releasing data helpful to
others in the field. In fact, even collaborating with others to publish
analyses of aggregated datasets results in multi-author publications for
which most participants receive little credit. The physics research
community had no choice but to work together when experiments became
exorbitantly expensive, and they have succeeded in changing the culture
so that collaborations have intrinsic value. This must happen in the
cancer susceptibility research community if important problems are to be
addressed.

**3. Develop a trans-NIH multiple-disease-oriented framework for
initiation of a very large-scale human population study, with multiple
ethnic groups, environmental exposures, and family histories. Ensure
widespread access to specimens, cell lines, and data.**

A central discussion point of the Cancer Susceptibility Think Tank was
the need to establish a million-person cohort from which standardized
samples and data could be obtained and shared among research groups
interested in complex diseases like cancer. The rationale and logistics
underlying such a large project, and its relationship to existing
cohorts, elicited extensive discussion. Among the considerations:

***Why a million-person cohort?***

Statisticians working on the proposal for the American Gene and
Environment Study concluded that a cohort of this size should be
adequate for analysis of gene-gene interactions of the type anticipated
to play a role in cancer susceptibility. An article by Francis Collins
in S*cience* mentioned the need for a large-scale longitudinal human
cohort, taking tissue samples pre- and post-disease, to make best use of
the technology -- not just after the disease has spread. Case control
studies typically yield biased information because they are based on the
surviving subset of people with that disease. Few studies have collected
long-term human tissue samples.

To design such a large population study is obviously challenging --
deciding what to analyze for and how to obtain a large enough dataset
that the signal-to-noise ratio is robust in the face of real human
variation. The difficulties are compounded by the high cost of the
undertaking, which necessitates that the cohort be applicable to
research on a broad spectrum of diseases. The studies must be very
long-term; thus the information and biological samples obtained and the
way in which they are stored must be carefully chosen to preclude their
obsolescence. Population characteristics can change dramatically in a
short time, as illustrated by variations in colorectal cancer rates with
environment and time.

***Building the cohort: Recruitment and Time-frame***

A trans-NIH multi-disease--oriented cohort should build on existing
cohort studies, and pilot novel strategies for database construction and
recruitment. It is critical to define the minimum data set needed, and
to make a *centralized data base* attractive to the cohort consortium.
Specimen resources should also be centralized, or at least federated, to
ensure access to them. It is also critical to put in place a mechanism
for obtaining available tissue when a participant develops cancer and
has a biopsy or surgery.

A million-person cohort will reach maximal utility within 5-10 years,
but recruitment should start as soon as possible. It is reasonable to
anticipate that technology will advance, and sequencing and genotyping
will become much easier to do. The 5-year goal should be based on
optimizing currently available technologies, while the 10-year goal
should assume the availability of high-throughput techniques applicable
to large populations.

***Specimen Collection, Standardization, Data Analysis, and
Accessibility***

When investing in such a large study, one important step is to determine
whether the current methods of defining diet and other environmental
exposures are adequate to capture all the information that may be of
value. Exposure measurements must be refined. It will also be critical
to know the genetics of cohort participants, from the germline to
somatic genetics and disease.

Specimen collection should include fresh tissue, benign as well as
malignant, associated with a minimum data set containing residency
history and ethnicity. A federated (virtual) facility would store and
distribute tissue and data. The "million cohort" database would be
accessible to all. Collection of tissue would be enabled, as would
international interactions. A grid system should be set up at NIH in
which all data from the "million cohort" would be included, with enough
computer power to do the modeling and analysis. The level of quality
control must give assurance of standardization and comparability of the
data.

**4. Exploit animal models for identification and validation of cancer
phenotypes**

**and susceptibility and resistance genes. Foster comparative genomic
and phenotypic studies of human and rodent (mouse and rat) cancer
susceptibility and resistance.**

Many laboratories use inbred and outbred mouse and rat strains to
disclose the genomic loci that are associated with complex traits, such
as tobacco carcinogenesis, addiction to alcohol, tobacco and other
drugs, diet-induced obesity, and hypertension, among others. There are
usually several dozen or more implicated loci, none of which has a major
effect by itself. Comparative genomics reveals that the majority of the
implicated loci are conserved across three species -- human, rat, and
mouse -- inspiring confidence that murine species can be informative for
the genetic and environmental determinants of human disease
susceptibility. The genomes for other species have also recently been
sequenced. In particular, championship bloodlines of dogs display
inherent susceptibility to a variety of cancers, and the exceptional
documentation of these bloodlines will enable cross-comparisons between
human populations and animals that share the same environment. An
expanding list of publications on the bases of susceptibility and
resistance to cancer and other diseases attests to the fact that
cross-species comparisons are not only useful, but also more facile,
approaches to defining complex traits.

Researchers are more optimistic about understanding genotype to
phenotype mapping using animal models rather than human models. It is
possible to generate animals in which the activity of several loci are
diminished or increased at the same time, and have a strain with a very
acute phenotype within a few months. An unbiased set of phenotyping
strategies can be applied to the strains to expose novel disease
manifestations that may be overlooked in human population studies. In
addition to unaltered inbred or outbred mice, mice that are genetically
altered to be sensitive or resistant to carcinogens are available. These
strains can be bred to others which differ in sensitivity to identify
which of the many contributing genes is dominant. Most wild mouse
strains have a low incidence of cancer, and studies of them indicate
that they have a number of dominant resistance genes. These are only a
few examples of existing mouse resources and approaches that can be
applied to the problem of understanding cancer susceptibility.

Several years ago, mouse models were designed to map simple,
single-gene--based traits; new mouse models are designed specifically
for analysis of complex traits and the contributions of environmental
effects. An integrative approach using newer mouse models to illuminate
results obtained with the million person cohort and to experimentally
model the resulting hypotheses would be particularly powerful. Even with
the best methods to model gene-gene or genetic-environment interactions,
statistical patterns that emerge from the mathematical models require
experimental validation in animals. This may require knockout mice for
evaluating multi-genotype variations. Not all the experimental
techniques are in place yet, nor have mice been engineered to examine
all the genotype interactions, but increased efforts to employ
integrative human/animal/computational strategies are likely to pay
substantial dividends in the study of human cancer susceptibility.

**5. Establish a trans-NIH initiative for the development of common
mouse resources for the analysis of genetic and environmental effects on
development of cancer and other complex trait diseases. Facilitate
multi-level, multi-lab studies of specimens after environmental
exposures for gene-environment interactions for the development of
computational models representing the complex genetic and non-genetic
networks associated with disease susceptibility and progression.**

Using wild-derived strains and out-bred populations, it is possible to
expand genetic variation far beyond that available in standard inbred
strains. Recombinant inbred (RI) lines are a powerful tool in the search
for susceptibility genes, and, even more importantly, for the conduct of
a systems biology approach to the correlation of genotype and phenotype.
A number of Think Tank participants expressed considerable enthusiasm
for the creation of a large, trans-NIH resource of RI strains to
facilitate study of cancer susceptibility and other similarly complex
genetic traits, such as diet-induced obesity. Several trans-NIH groups
are evaluating common mouse resources to understand complex human
diseases, and there are international efforts underway to coordinate the
development and deployment of these resources. The precise nature of
common mouse resources for the analysis of complex gene-environment
interactions in human diseases still requires consensus-building within
the relevant research community. These efforts are the subjects of a
series of workshops currently in development.

**6. Radically modify training programs to provide immersive
interdisciplinary learning** **environments for biologists,
mathematicians, computer scientists,** **statisticians, epidemiologists,
and clinical investigators.**

The field of cancer susceptibility needs investigators who can
understand and apply high-level computational and modeling techniques in
analyzing multiple genetic and environmental interactions. Think Tank
participants identified several approaches for overcoming this problem.

***Develop cross-training, interdisciplinary programs***

Cross-training in biology and computational science is challenging. Most
of the participants expressed a preference for educating scientists with
programming or statistical genetics or mathematical modeling backgrounds
about biology, rather than teaching biologists the math and
computational skills. Thus, it is important to identify students with
good quantitative skills at the undergraduate level who are comfortable
with statistical concepts so that mathematics is a fundamental component
of their broad education. It is also important to educate biologists and
epidemiologists how to generate and apply models. Too often, data that
are analyzed are not translated into a model, or the models that are
created only verify the original assumptions and add nothing new.

A major change is needed in graduate training to prepare scientists in
quantitative and biological areas, but successful prototypes exist. For
example, an innovative Ph.D. program exists for mathematicians,
physicists and computer scientists to learn biology as well as use their
technical skills, and for biologists and chemists to spend most of their
time learning how to model. The field of Neuroscience is unusual in that
it allows researchers to return at any stage of their career for
cross-disciplinary training. Similar opportunities would be of value in
cancer research.

***Provide grant support for interdisciplinary education***

Most NIH training grant programs limit the opportunities for students to
pursue interdisciplinary opportunities or learn computational methods,
yet students should be rewarded for seeking training in other areas.
Competitive grants and awards designated for that purpose would
encourage pursuit of interdisciplinary education. A few NIH grants,
awarded to an institution or an individual, do require interdisciplinary
training, with two mentors and work in two labs in different areas.
Expansion of these programs is strongly encouraged, could be done
immediately, and would have an impact within ten years. Anything that
increases cross-disciplinary training is an opportunity with a good
pay-off. Such training could be encouraged by NCI/NIH and implemented
without additional funds -- just reconfiguration of the present
priorities.

***Support summer programs and/or workshops***

Although interdisciplinary training is hard to find within an
institution, some institutions convene special summer programs. NCI or
NIH could support a program of intensive lectures to introduce young
people to concepts such as model building. A program might involve 50-75
students, staff support, and facilities that can deal with animals,
cells, human material, and computational materials. Students could be
familiarized with enough mathematics to feel comfortable, and high-end
lecturers could be brought in, as is the practice at courses at Woods
Hole and Cold Spring Harbor. Courses of this type have not been
implemented to provide interdisciplinary training for mathematicians and
cancer biologists. Another possibility for productive interaction would
be a genetic analysis workshop, in which the participants could work
with a shared dataset and apply their own methods to analyze the data.

***Increase access to hardware and large-scale resources***

Students occasionally have problems with access to computers, but their
main problem is accessing higher-level analyses. Students who are unable
to parallelize their data may take months to learn the algorithms, and
are unable to perform analyses on the higher power machines. In another
three years, there will be more computers with unusual architectures;
there is an immediate need to train biomedical scientists to use these
new computing technologies and to provide more parallelization training
so the next generation of scientists will be able to move into the new
systems. Grants to purchase instrumentation to be used for training
would encourage institutions to provide training.

***Encourage collaborative career development***

At many universities, junior faculty members who publish with other
people receive less credit than for a solo publication; this strongly
discourages collaborations and interactions across disciplines. Yet
within academia, there are a few models like Biostatistics, in which
people build careers with collaborative studies and still advance the
core of their own discipline. The work-style of NIH-supported individual
investigator communities is generally counter to the kind of
collaborations needed to make real progress. NCI should investigate and
implement incentives of recognition as well as financial rewards to
encourage necessary culture change.

Biomedical scientists tend to utilize computer scientists as
technicians, so computational scientists hesitate to get involved in
collaborations in this area. When they are recognized as a critical
intellectual partner, computer scientists contribute novel ideas to the
design and execution of the project, and analysis of the outcome. It
also helps to have a critical mass of computational people involved in
biological research, so that they have a community to interact with on
theoretical and development issues.

**Specific Recommendations for the NCI:**

Realizing that the goal for cancer susceptibility research set out in
the Executive Summary will be possible only with advances in basic
knowledge about cancer and genetic variation, and the introduction of
new tools and approaches, the following are recommended.

***Requirements to achieve the goal:***

-   Integrate studies of experimental model systems, human populations,
    and computational models of molecular signatures in normal and
    dysregulated states relevant to particular features of cancer
    susceptibility.

-   Improve cancer-related phenotyping (i.e., the analysis of molecular
    and systemic changes in cancer progression) by applying emerging
    sensor, imaging, nano-and molecular technologies to individual
    humans and animals, biological specimens, cells, extracellular
    matrix components, and cell-cell interactions

-   Identify sources of individual variation in cancer-related
    biological processes, both genetic and non-genetic, and model the
    genetic-environmental and stochastic interactions.

-   Develop and apply technologies to investigate genetic variation
    broadly, including alleles, SNPs, haplotypes, and epigenetic
    alterations of nuclear and mitochondrial genomes, miRNA, highly
    repetitive sequences, transposons, gene copy number polymorphisms
    and other evolutionary molecular features.

***Specific recommendations:***

-   Given the scale of datasets and complex biological systems, foster
    the creation of new computational, mathematical, and statistical
    models and analytical tools that embrace, rather than simplify or
    ignore the complexity of variation in susceptibility and resistance
    to cancers. Support large-scale coordination of studies with
    calibration, interoperability, data federation, and standardization
    of data formats, natural language processing tools, and dataset
    query tools.

-   Create a common and widely available repository with existing
    resources of well-selected, maintained, and annotated human
    biological specimens (immortalized lymphoblastoid cell lines, serum,
    plasma) for genetic, epigenetic, proteomic, and metabolomic analyses
    of early-onset cancer cases, including those with positive family
    histories. Create common databases. Develop a corresponding resource
    from a special elderly subpopulation to study resistance to cancer
    development in individuals with high lifetime exposure to
    carcinogens. Require data deposition in freely accessible databases,
    and resource sharing of repository materials.

-   Develop a trans-NIH multiple-disease--oriented framework for
    initiation of a very large-scale human population study, with
    multiple ethnic groups, environmental exposures, and family
    histories. Ensure widespread access to specimens, cell lines, and
    data.

-   Exploit existing animal models for identification and validation of
    cancer phenotypes and susceptibility and resistance genes. Foster
    comparative genomic and phenotypic studies of human and rodent
    (mouse and rat) and other mammal cancer susceptibility and
    resistance.

-   Establish a trans-NIH initiative for the development of common mouse
    resources for the analysis of genetic and environmental effects on
    development of cancer and other complex trait diseases. Facilitate
    multi-level, multi-laboratory studies of specimens after
    environmental exposures for gene-environment interactions for the
    development of computational models representing the complex genetic
    and non-genetic networks associated with disease susceptibility and
    progression.

-   Radically modify training programs to provide immersive
    interdisciplinary learning environments for biologists,
    mathematicians, computer scientists, statisticians, epidemiologists,
    and clinical investigators

    **Tumor Immunology Think Tank**

    **January 22-24, 2003**

    **PARTICIPANT ROSTER**

    **James P. Allison, Ph.D.**

    Co-organizer

Professor and Co-chair

Howard Hughes Medical Institute

University of California, Berkeley

401 LSA

Berkeley, CA 94720-3200

Tel: 510-643-6012

Fax: 510-643-5692

Email: <jallison@uclink.berkeley.edu>

**Drew Pardoll, M.D., Ph.D.**

Co-organizer

Professor, Department of Oncology

Johns Hopkins University School

of Medicine

1650 Orleans Street

Bunting Blaustein Building #440

Baltimore, MD 21231

Tel: 410-955-7866

Fax: 410-614-0549

Email: <dmpardol@jhmi.edu>

**Paul M. Allen, Ph.D.**

R.L. Kroc Professor

Department of Pathology

Washington University

School of Medicine

660 South Euclid

Campus Box 8118

St. Louis, MO 63110-1010

Tel: 314-362-8758

Fax: 314-362-8888

Email: <allen@immunology.wustl.edu>

**Jacques Banchereau, Ph.D.**

Director, Baylor Institute for

Immunologic Research

3434 Live Oak, Suite 205

Dallas, TX 75204-6134

Tel: 214-820-7450

Fax: 214-820-4813

Email: <jacquesb@baylorhealth.edu>

**Jay A. Berzofsky, M.D., Ph.D.**

Chief, Immunogenetics and Vaccine

Research Section

National Cancer Institute, NIH

Building 10, Room 6B12

Bethesda, MD 20892-1578

Tel: 301-496-6874

Fax: 301-496-9956

Email: <berzofsk@helix.nih.gov>

**Harvey Cantor, M.D.**

Professor and Chair, Department of

Immunology and AIDS

Dana Farber Cancer Institute

Harvard Medical School

44 Binney Street

Boston, MA 02115

Tel: 617-632-3348

Fax: 617-632-4630

Email: <Harvey_cantor@dfci.harvard.edu>

**Lieping Chen, M.D., Ph.D.**

Professor, Department of Immunology

Mayo Medical School

Mayo Clinic

200 First Street, SW

Guggenheim 342B

Rochester, MN 55905

Tel: 507-538-0013

Fax: 507-284-1637

Email: <chen.lieping@mayo.edu>

**Stephen P. Creekmore, Ph.D.**

Biological Resources Branch, DCTD

National Cancer Institute

NCI-Frederick, Bldg. 1052, Rm. 251

Frederick, MD 21702-1201

Tel: 301-846-1100

Fax: 301-846-5429

Email: <sc63a@nih.gov>

**Glenn Dranoff, M.D.**

Dana Farber Cancer Institute

Dana 510E

44 Binney Street

Boston, MA 02115

Tel: 617-632-5051

Fax: 617-632-5167

Email: <glenn_dranoff@dfci.harvard.edu>

**Margaret Foti, Ph.D.**

Chief Executive Officer

American Association for Cancer Research

Public Ledger Building, Suite 826

150 S. Independence Mall West

Philadelphia, PA 19106-3483

Tel: 215-440-9300, ext. 210

Fax: 215-440-9322

Email: <foti@aacr.org>

**Ronald N. Germain, M.D., Ph.D.**

Chief, Lymphocyte Biology Section

Laboratory of Immunology

NIAID, NIH

Building 10, Room 11N311

Bethesda, MD 20892-1892

Tel: 301-496-1904

Fax: 301-496-0222

Email: <rgermain@nih.gov>

**Eli Gilboa, Ph.D.**

Professor, Department of Experimental Surgery

Duke University Medical Center

DUMC 2601

401D MSR Building

Durham, NC 27710

Tel: 919-684-6465

Fax: 919-668-2587

Email: <e.gilboa@cgct.duke.edu>

**Philip D. Greenberg, M.D.**

Professor, Departments of Medicine and Immunology

University of Washington

Room BB1325, Health Sciences Building

Box 356527

Seattle, WA 98195-6527

Tel: 206-543-8306

Fax: 206-685-3128

Email: <pgreen@u.washington.edu>

**Ronald E. Gress, M.D.**

Senior Investigator

NCI, NIH

Building 10, Room 4B17

Bethesda, MD 20892-0001

Tel: 301-496-5461

Fax: 301-496-0887

Email: <rg26g@nih.gov>

**Lauren G. Gross**

Director of Public Policy and Government Affairs

The American Association of Immunologists

9650 Rockville Pike

Bethesda, MD 20814

Tel: 301-530-7178

Fax: 301-571-1816

Email: <lgross@aai.faseb.org>

**Douglas Hanahan, Ph.D.**

Professor, Department of Biochemistry and Biophysics

University of California San Francisco

513 Parnassus Avenue

HSW 1090

San Francisco, CA 94143

Tel: 415-476-9209

Fax: 415-502-1447

Email: <dh@biochem.ucsf.edu>

**Toby T. Hecht, Ph.D.**

Biological Resources Branch, DCTD

National Cancer Institute

NCI-Frederick, Bldg. 1052, Rm. 247

Frederick, MD 21702-1201

Tel: 301-846-5692

Fax: 301-846-5429

Email: <th30h@nih.gov>

**Michele Hogan, Ph.D.**

Executive Director

The American Association of Immunologists

9650 Rockville Pike

Bethesda, MD 20814

Tel: 301-530-7178

Fax: 301-571-1816

Email: <mmhogan@aai.faseb.org>

**Elizabeth M. Jaffee, M.D.**

Associate Professor, Department of Oncology

Johns Hopkins Hospital

1650 Orleans Street

Bunting Blaustein Building, Room 4M07

Baltimore, MD 21231

Tel: 410-955-2957

Fax: 410-614-8216

Email: <ejaffee@jhmi.edu>

**Hyam I. Levitsky, M.D.**

Prof. of Oncology, Medicine & Urology

Johns Hopkins Univ. Sch. of Medicine

Sidney Kimmel Comprehensive Cancer Center

4M51 Bunting Blaustein Cancer

Research Building

1650 Orleans Street

Baltimore, MD 21231

Tel: 410 614-0552

Fax: 410-614-9705

Email: [hy@jhmi.edu](mailto:hy@welchlink.welch.jhu.edu)

**Philippa Marrack, Ph.D.**

Investigator, Department of Immunology, HHMI

National Jewish Medical and Research Center

1400 Jackson Street

Denver, CO 80206-2761

Tel: 303-398-1322

Fax: 303-398-1396

Email: <marrackp@njc.org>

**Cornelis J. M. Melief, M.D., Ph.D.**

Department of Immunohematology and Blood Bank

University Hospital

Building 1, E3-Q

P.O. Box 9600

2300 RC Leiden, The Netherlands

Tel: +31 (71) 526-3800

Fax: +31 (71) 521-6751

Email: <cmelief@lumc.nl>

**Pamela S. Ohashi, Ph.D.**

Senior Scientist and Professor

Division of Cellular & Molecular

Biology

Ontario Cancer Institute

University Health Network

610 University Avenue

Toronto, Ontario M5G 2M9 Canada

Tel: 416-946-4501, ext.5470

Fax: 416-946-2086

Email: <pohashi@oci.utoronto.ca>

**Richard J. O'Reilly, M.D.**

Professor, Department of Pediatrics

Memorial Sloan-Kettering Cancer Center

1275 York Avenue

New York, NY 10021

Tel: 212-639-5957

Fax: 212-717-3447

Email: <oreillyr@mskcc.org>

**Steven A. Rosenberg, M.D., Ph.D.**

Chief, Surgery Branch

NCI, NIH

Building 10, Room 2B42

Bethesda, MD 20892-1502

Tel: 301-496-4164

Fax: 301-402-1738

Email: <sr78a@nih.gov>

**David A. Scheinberg, M.D., Ph.D.**

Chief, Leukemia Service

Department of Medicine

Memorial Sloan-Kettering Cancer Center

1275 York Avenue

New York, NY 10021

Tel: 212-639-5010

Fax: 212-717-3068

Email: <d-scheinberg@ski.mskcc.org>

**Robert D. Schreiber, Ph.D.**

Professor, Departments of Pathology and Immunology

Washington University School of Medicine

660 South Euclid Avenue

Box 8118

St. Louis, MO 63110-1093

Tel: 314-362-8747

Fax: 314-747-4888

Email: <Schreiber@immunology.wustl.edu>

**Linda A. Sherman, Ph.D.**

Professor, Department of Immunology

IMM-15

Scripps Research Institute

10550 North Torrey Pines Road

La Jolla, CA 92037-1092

Tel: 858-784-8052

Fax: 858-784-8298

Email: <lsherman@scripps.edu>

**Alfred Singer, M.D.**

Chief, Experimental Immunology Branch

NCI, NIH

Building 10, Room 4B36

Bethesda, MD 20892-1360

Tel: 301-496-5461

Fax: 301-496-0887

Email: <singera@mail.nih.gov>

**Howard Streicher, M.D.**

Senior Investigator

NCI/CTEP

6130 Executive Boulevard, Suite 7126

Rockville, MD 20892

Tel: 301-435-9129

Fax: 301-402-0428

Email: <streicherh@ctep.nci.nih.gov>

**Craig B. Thompson, M.D.**

Scientific Director, Abramson Family Cancer Research Institute

University of Pennsylvania

421 Curie Boulevard

451 BRBII/III

Philadelphia, PA 19104-6160

Tel: 215-746-5515

Fax: 215-746-5511

Email: [craig@mail.med.upenn.edu](mailto:drt@mail.med.upenn.edu)

**Harald Von Boehmer, M.D., Ph.D.**

Professor, Department of Pathology

Dana Farber Cancer Institute

44 Binney Street

Smith 736

Boston, MA 02115

Tel: 617-632-6880

Fax: 617-632-6881

Email: <harald_von_boehmer@dfci.harvard.edu>

**Thomas A. Waldmann, M.D.**

Chief, Metabolism Branch

NCI, NIH

Building 10, Room 4N115

Bethesda, MD 20892

Tel: 301-496-6653

Fax: 301-496-9956

Email: <tw26v@nih.gov>

**Irving Weissman, M.D.**

Professor, Department of Pathology

Stanford University

School of Medicine

B257 Beckman Center

Stanford, CA 94305-5323

Tel: 650-723-6520

Fax: 650-723-4034

Email: <irv@stanford.edu>

**Tumor Microenvironment Think Tank**

**November 5-7, 2003**

**PARTICIPANT ROSTER**

**Joan S. Brugge, Ph.D., Co-Chair**

Professor of Cell Biology\
Harvard Medical School\
240 Longwood Ave\
Boston, MA 02115\
Tel: 617-432-3974\
Fax: 617-432-3969\
Email: <joan_brugge@hms.harvard.edu>

**G. David Roodman, M.D., Ph.D., Co-Chair**

Professor, Department of Medicine

Vice Chair of Research

University of Pittsburgh

Kaufman Medical Building

3471 Fifth Avenue, Suite 601

Pittsburgh, PA 15213

Tel: 412-692-4438

Fax: 412-692-4144

Email: [roodmangd@msx.upmc.edu](mailto:roodmangd@upmc.edu)

**Kenneth C. Anderson, M.D.**

Kraft Family Professor of Medicine

Department of Adult Oncology

Harvard Medical School

Dana-Farber Cancer Institute

44 Binney Street

Boston, MA 02115

Tel: 617-632-2144

Fax: 617-632-2140

Email: <kenneth_anderson@dfci.harvard.edu>

**Ravi Bhatia, M.D.**

Director, Stem Cell Biology Program

City of Hope Comprehensive Cancer Center

15000 E. Duarte Road

Duarte, CA 91010

Tel: 626-359-8111, Ext. 63974

Fax: 626-301-8973

Email: <rbhatia@coh.org>

**Mina J. Bissell, Ph.D.**

Distinguished Scientist\
Lawrence Berkeley National Laboratory\
One Cyclotron Road

Mailstop 83, Room 101\
Berkeley, CA 94720-8260

Tel: 510-486-4365\
Fax:  510-486-5586\
Email: <MJBissell@lbl.gov>

**Patrick O. Brown, M.D., Ph.D.**

Investigator/Professor

Howard Hughes Medical Institute

Stanford University Department

of Biochemistry

Beckman Center

279 Campus Dr, Room B439

Stanford, CA 94605-5307

Tel: 650-723-0005

Fax: 650-725-7811

Email: <pbrown@cmgm.stanford.edu>

**Dario Campana, M.D., Ph.D.**

Member and Professor

Department of Hematology and Oncology

St. Jude Children's Research Hospital

332 N. Lauderdale

Memphis, TN 38105

Tel: 901-495-2528

Fax: 901-495-3749

Email: <dario.campana@stjude.org>

**Yong S. Choi, M.D., Ph.D.**

Director and Professor

Department of Cellular Immunology

Ochsner Clinic Foundation

1516 Jefferson Highway

New Orleans, LA 70121

Tel: 504-842-3035

Fax: 504-842-3381

Email: <ychoi@ochsner.org>

**John Condeelis, Ph.D.**\
Professor and Co-Chair

Department of Anatomy and

Structural Biology

Albert Einstein College of Medicine\
1300 Morris Park Avenue\
Forchheimer Building - Room 628\
Bronx, New York 10461\
Tel: 718-430-4068\
Fax: 718-430-8996\
Email: <condeeli@aecom.yu.edu>

**Lisa M. Coussens, Ph.D.**

Assistant Professor, In Residence

Department of Pathology

University of California, San Francisco

2340 Sutter Street, Box 0875

San Francisco, CA 94143-0875

Tel: 415-502-6378

Fax: 415-514-0878

Email: <coussens@cc.ucsf.edu>

**William S. Dalton, M.D., Ph.D.**

CEO and Center Director

H. Lee Moffitt Cancer Center

and Research Institute

12902 Magnolia Drive

Tampa, FL 33612-9497

Tel: 813-615-4261

Fax: 813-615-4258

Email: <dalton@moffitt.usf.edu>

**Joshua Epstein, D.Sc.**

Professor of Medicine

Myeloma Institute for Research

and Therapy

4301 West Markham, #776

Little Rock, AR 72205

Tel: 501-686-8250

Fax: 501-686-6442

Email: [epsteinjoshua@uams.edu](mailto:epsteinjoshua@exchange.uams.edu)

**Isaiah J. Fidler, D.V.M., Ph.D.**

Professor and Department Chairman

Department of Cancer Biology-173

University of Texas M.D. Anderson

Cancer Center

1515 Holcombe Boulevard

Houston, TX 77030

Tel: 713-792-8580

Fax: 713-792-8747

Email: <ifidler@mdanderson.org>

**Mary J.C. Hendrix, Ph.D.**

Professor and Head

Department of Anatomy and Cell Biology

The University of Iowa

1-100 Bowen Science Building

Iowa City, IA  52242-1009

Tel:  319-335-7755

Fax:  319-335-7770

Email: <mary-hendrix@uiowa.edu>

**Meenhard Herlyn, D.V.M., D.Sc.**

Professor and Chair

Molecular and Cellular Oncogenesis

Program

The Wistar Institute

3601 Spruce Street, Room 489

Philadelphia, PA 19104

Tel: 215-898-3950

Fax: 215-898-0980

Email: <herlynm@wistar.upenn.edu>

**Richard S. Larson, M.D., Ph.D.**

Associate Professor

Department of Pathology

Cancer Research Facility

University of New Mexico

2325 Camino De Salud NE, Rm. 223

Albuquerque, NM 87121

Tel: 505-272-9762

Fax: 505-272-5186

Email: <rlarson@salud.unm.edu>

**Lynn M. Matrisian, Ph.D.**

Professor and Chair

Department of Cancer Biology

Vanderbilt University Medical Center

2220 Pierce Avenue/771 Preston Building

Nashville, TN  37232-6840

Tel: 615-322-0375

Fax: 615-936-2911

Email: <lynn.matrisian@vanderbilt.edu>

**Anil K. Rustgi, M.D.**

T. Grier Miller Professor of Medicine

and Genetics

Chief of Gastroenterology

University of Pennsylvania

415 Curie Blvd., 6 CRB

Philadelphia, PA 19104

Tel: 215-573-5412

Fax: 215-812-1320

Email: <anil2@mail.med.upenn.edu>

**Hans Schreiber, M.D., Ph.D.**

Professor

Department of Pathology

University of Chicago

5841 S. Maryland Avenue, MC 1089

Chicago, IL 60637

Tel: 773-702-9204

Fax: 773-702-9224

E-mail: <hszz@midway.uchicago.edu>

**George D. Yancopoulos, M.D., Ph.D.\
**President of Research Laboratories

CSO, Regeneron Pharmaceuticals, Inc.\
777 Old Saw Mill River Road\
Tarrytown, NY  10591-6707\
Tel: 914-345-7423\
Fax: 914-345-7650\
E-Mail: <george@regeneron.com>

**Bruce R. Zetter, Ph.D.**

Vice President for Research

Charles Nowiszewski Professor of

Cancer Biology

Harvard Medical School

Children's Hospital Boston

300 Longwood Avenue, Enders 1077

Boston, MA 02115

Tel: 617-355-1864

Fax: 617-730-0232

Email: <bruce.zetter@childrens.harvard.edu>

**Tumor Stem Cells and the Genetic Basis of Self-Renewal Think Tank**

**March 24-26, 2004**

**PARTICIPANT ROSTER**

**Michael Clarke, M.D.**

Professor

Departments of Internal Medicine and

Cell and Developmental Biology

University of Michigan

4310 CCGC

1500 E. Medical Center Drive

Ann Arbor, MI 48109-0936

Tel: 734-764-8195

Fax: 734-647-9654

Email: <mclarke@umich.edu>

**Robert Coffey, M.D.**

Professor of Medicine and Cell and

Developmental Biology

Department of Gastroenterology

Vanderbilt University

4140 MRB III

465 21^st^ Avenue South, Suite 7140

Nashville, TN 37232-2279

Tel: 615-346-6228

Fax: 615- 343-1591

Email: <robert.coffey@vanderbilt.edu>

**John Dick, Ph.D.**

Canada Research Chair in Stem

Cell Biology

Toronto General Research Institute

University Health Network

620 University Avenue, Suite 700

Toronto, Ontario M5G 2C1

Canada

Tel: 416-946-6549

Fax: 416-946-6589

Email: <jdick@uhnres.utoronto.ca>

**Peter Dirks, M.D., Ph.D.**

Assistant Professor

Developmental Biology Program

Hospital for Sick Children

555 University Avenue

Toronto, Ontario M5G 1X8

Canada

Tel: 416-813-6426

Fax: 416-813-4975

Email: <peter.dirks@sickkids.ca>

**William Dove, Ph.D.**

Professor, Oncology and Medical Genetics

University of Wisconsin

1400 University Avenue

Madison, WI 53706-1599

Tel: 608-262-4977

Fax: 608-262-2824

Email: <dove@oncology.wisc.edu>

**Rudolf Jaenisch, M.D.**

Professor of Biology

Member, Whitehead Institute for

Biomedical Research

Massachusetts Institute of Technology

Nine Cambridge Center

Cambridge, MA 02142

Tel: 617-258-5186

Fax: 617-258-6505

Email: <jaenisch@wi.mit.edu>

**Richard Jones, M.D.**

Professor of Oncology

Sidney Kimmel Comprehensive

Cancer Center

Johns Hopkins University

Bunting-Blaustein Cancer Research

Building, Room 207

1650 Orleans Street

Baltimore, MD 21231-1000

Tel: 410-955-2006

Fax: 410-502-7213

Email: <rjjones@jhmi.edu>

**Craig Jordan, Ph.D.**

Associate Professor

Department of Medicine

University of Rochester Medical Center

601 Elmwood Avenue, Box 703

Rochester, NY 14642

Tel: 585-275-6339

Fax: 708-575-3785

Email: <craig_jordan@urmc.rochester.edu>

**Ihor Lemischka, Ph.D.**

Professor

Department of Molecular Biology

Lewis Thomas Laboratory

Princeton University

Washington Road

Princeton, NJ 08544

Tel: 609-258-2838

Fax: 609-258-2759

Email:

<ilemischka@molbio.princeton.edu>

**Roel Nusse, Ph.D.**

Professor

Department of Developmental Biology

Howard Hughes Medical Institute

Beckman Center

Stanford University

Stanford, CA 94305

Tel: 650-723-7769

Fax: 650-723-1399

Email: <rnusse@cmgm.stanford.edu>

**Luis Parada, Ph.D.**

Director

Center for Developmental Biology

University of Texas Southwestern Medical

Center

5323 Harry Hines Boulevard

Dallas, TX 75390-9133

Tel: 214-648-1822

Fax: 214-648-1960

Email: <luis.parada@utsouthwestern.edu>

**Guy Sauvageau, M.D., Ph.D.**

Director, Laboratory of Molecular Genetics

of Stem Cells

Faculty of Medicine

Cancer and Immunology Institute

Université de Montréal

Pavillon Principal, T439 C.P. 6128

Succ. Centre-Ville

Montréal, Québec, H3C 3J7

Canada

Tel: 514-343-7134

Fax: 514-343-7379

Email: <guy.sauvageau@umontreal.ca>

**David Scadden, M.D.**

Director, Center for Regenerative Medicine

and Technology

Co-Director, Harvard Stem Cell Institute

Massachusetts General Hospital

149 13th Street, Room 5212D

Boston, MA 02129

Tel: 617-726-5615

Fax: 617-726-4691

Email:

<scadden.david@mgh.harvard.edu>

**Charles Sherr, M.D., Ph.D.**

Investigator, Howard Hughes Medical

Institute

Genetics and Tumor Cell Biology

St. Jude Children\'s Research Hospital

332 North Lauderdale

Memphis, TN 38105

Tel: 901-495-3505

Fax: 901-495-2381

Email: <sherr@stjude.org>

**Austin Smith, Ph.D.**

Professor

Institute for Stem Cell Research

University of Edinburgh

King\'s Buildings, West Mains Road

Edinburgh EH9 3JQ

Scotland, United Kingdom

Tel: 44-131-650-5828

Fax: 44-131-650-7773

Email: <austin.smith@ed.ac.uk>

**Gil Smith, Ph.D.**

Senior Investigator

Mammary Biology and Tumorigenesis

Laboratory, CCR

National Cancer Institute

Building 10, Room 5B56

Bethesda, MD 20892

Phone: 301-496-2385

Fax: 301-480-1790

Email: <gs4d@nih.gov>

**Robert Weinberg, Ph.D.**

Member

Whitehead Institute for Biomedical

Research

Department of Biology

Massachusetts Institute of Technology

9 Cambridge Center

Cambridge, MA 02142

Tel: 617-258-5159

Fax: 617-258-5213

Email: <weinberg@wi.mit.edu>

**Irv Weismann, M.D., Ph.D.**

Director of the Institute of Cancer/Stem Cell

Biology and Medicine, Professor of

Pathology and Developmental Biology

Department of Pathology

Stanford University Medical Center

Beckman Room 257

279 Campus Drive

Stanford, CA 94305-5323

Tel: 650-723-6520

Fax: 650-723-4034

Email: <irv@stanford.edu>

**Max Wicha, M.D.**

Director, Comprehensive Cancer Center

Professor, Internal Medicine

University of Michigan

6302 CCGC, Box 0942

1500 East Medical Center Drive

Ann Arbor, MI 48109-0942

Tel: 734-936-1831

Fax: 734-615-3947

Email: <mwicha@umich.edu>

**Hong Wu, M.D., Ph.D.**

Associate Professor

Molecular and Medical Pharmacology

University of California Los Angeles School of Medicine

650 CE Young Drive South

Los Angeles, CA 90095-1735

Tel: 310-825-5160

Fax: 310-267-0242

Email: <hwu@mednet.ucla.edu>

**Cell Decision in Response to DNA Damage:**

**Survival vs. Programmed Cell Death Think Tank**

**April 13-15, 2004**

**PARTICIPANT ROSTER**

**Jiri Bartek, M.D., Ph.D.**

Head, Department of Cell Cycle and Cancer Institute of Cancer Biology

Danish Cancer Society

Strandboulevarden 49

DK-2100 Copenhagen

Denmark

Tel: 45-3525-7357

Fax: 45-3525-7721

Email: <bartek@biobase.dk>

**Eric Brown, Ph.D.**

Assistant Professor

Department of Cancer Biology

University of Pennsylvania

BRB II/III, Room 510

421 Curie Boulevard

Philadelphia, PA 19104-6160

Tel: 215-746-2805

Fax: 215-573-2486

Email: <brownej@mail.med.upenn.edu>

**Gerard Evan, Ph.D.**

Gerson and Barbara Bass Baker

Distinguished Professor of Cancer Biology, Cellular and Molecular

Pharmacology

Cancer Research Institute

University of California, San Francicso

2340 Sutter Street, Campus Box 0875

San Francisco, Ca 94115

Tel: 415-514-0438

Fax: 415-514-0878

Email: <gevan@cc.ucsf.edu>

**David Fisher, M.D., Ph.D.**

Associate Professor

Department of Pediatric Oncology

Dana-Farber Cancer Institute

44 Binney Street, D630

Boston, MA 02115

Tel: 617-632-4916

Fax: 617-632-2085

Email: <david_fisher@dfci.harvard.edu>

**Albert Fornace, Jr., M.D.**

Chief, Gene Response Section

Basic Research Laboratory, CCR

National Cancer Institute

Building 37, Room 6144

Bethesda, MD 20892-4255

Tel: 301-402-0744

Fax: 301-480-1946

Email: <furnace@nih.gov>

**Zvi Fuks, M.D.**

Deputy Physician-in-Chief for Planning

Department of Radiation Oncology

Memorial Sloan-Kettering Cancer Center

1275 York Avenue

New York, NY 10021

Tel: 212-639-5868

Fax: 212-794-3188

Email: <fuksz@mskcc.org>

**Amato Giaccia, Ph.D.**

Associate Professor

Department of Radiation Oncology

Division of Radiation and Cancer Biology

Stanford University School of Medicine

CCSR-South, Room 1255\
269 Campus Drive

Stanford, CA 94305-5152

Tel: 650-723-7366

Fax: 650-723-7382

Email: <giaccia@stanford.edu>

**Douglas Green, Ph.D.**

Head, Division of Cellular Immunology

La Jolla Institute for Allergy and Immunology

10355 Science Center Drive

San Diego, CA 92121

Tel: 858-558-3500

Fax: 858-558-3594

Email: <dgreen5240@aol.com>

**Andrei Gudkov, Ph.D., D.Sci.**

Chairman, Department of Molecular Biology

Lerner Research Institute/Cleveland Clinic

9500 Euclid Avenue, NC20

Cleveland, OH 44195

Tel: 216-445-1205

Fax: 216-444-0512

Email: <gudkov@ccf.org>

**Thanos Halazonetis, D.D.S., Ph.D.**

Associate Professor

Department of Molecular Genetics

Wistar Institute

3601 Spruce Street

Philadelphia, PA 19104-4268

Phone: (215) 898-3789

Fax: (215) 573-9271

Email: halazonetis@wistar.upenn.edu

**Stephen Kowalczykowski, Ph.D.**

Professor of Microbiology and Director\
Center for Genetics and Development

University of California, Davis

Microbiology-310 Briggs Hall

One Shields Avenue

Davis, CA 95616

Tel: 530-752-5938

Fax: 530-752-5939

Email:

<sckowalczykowski@ucdavis.edu>

**Yuri Lazebnik, Ph.D.**

Professor

Cold Spring Harbor Laboratory

One Bungtown Road

Cold Spring Harbor, NY 11724

Tel: 516-367-8363

Fax: 516-367-8461

Email: <lazebnik@cshl.edu>

**Scott Lowe, Ph.D.**

Professor and Deputy Director\
of Cancer Center

Cold Spring Harbor Laboratory

One Bungtown Road

Cold Spring Harbor, NY 11724

Tel: 516-367-8406

Fax: 516-367-8454

Email: <lowe@cshl.edu>

**Peter McKinnon, Ph.D.**

Associate Member

Department of Genetics and Tumor\
Cell Biology

St. Jude Children's Research Hospital

332 N. Lauderdale Street

Memphis, TN 38105-2794

Tel: 901-495-2700

Fax: 901-526-2907

Email: <peter.mckinnon@stjude.org>

**Raymond Monnat, Jr., M.D.**

Professor, Departments of Pathology\
and Genetics/Genome Sciences

University of Washington

Seattle, WA 98195-7705

Tel: 206-616-7392

Fax: 206-543-3967

Email: <monnat@u.washington.edu>

**John Petrini, Ph.D.**

Member of Molecular Biology Department

Division of Chromosome Biologyuj

Memorial Sloan-Kettering Cancer Center

1275 York Avenue, RRL 901B

New York, NY 10021

Tel: 212-639-2927

Fax: 646-422-2062

Email: <petrinij@mskcc.org>

**Neal Rosen, M.D., Ph.D.**

Member

Department of Medicine and Cell Biology

Memorial Sloan-Kettering Cancer Center

1275 York Avenue

New York, NY 10021

Tel: 212-639-2369

Fax: 212-717-3627

Email: <rosenn@mskcc.org>

**Lorraine Symington, Ph.D.**

Professor

Department of Microbiology

Columbia University Medical Center

701 West 168th Street

New York, NY 10032

Tel: 212-305-4793

Fax: 212-305-1741

Email: <lss5@columbia.edu>

**Craig Thompson, M.D.**

Scientific Director

Abramson Family Cancer Research Institute

University of Pennsylvania

Room 450 BRB II/III

421 Curie Boulevard

Philadelphia, PA 19104-6160

Tel: 215-746-5515

Fax: 215-746-5511

Email: <craig@mail.med.upenn.edu>

**Jean Wang, Ph.D.**

Professor, Division of Biology

Department of Molecular Biology

University of California, San Diego

9500 Gilman Drive

La Jolla, CA 92093-0322

Tel: 858-534-6253

Fax: 858-822-2002

Email: <jywang@ucsd.edu>

**Michael Yaffe, M.D., Ph.D.**

Associate Professor

Center for Cancer Research

Massachusetts Institute of Technology

77 Massachusetts Avenue, Building E18-580

Cambridge, MA 02139

Tel: 617-452-2442

Fax: 617-452-4978

Email: <myaffe@mit.edu>

**Cancer Etiology: Role of Exogenous and Endogenous Chemicals**

**April 15-17, 2004**

**PARTICIPANT ROSTER**

**Steven Belinsky, Ph.D.**

Director of Lung Cancer Program

Lovelace Respiratory Research Institute

2425 Ridgecrest Drive, S.E.

Albuquerque, NM 87108

Tel: 505-348-9465

Fax: 505-348-4990

Email: <sbelinsk@lrri.org>

**L. Michelle Bennett, Ph.D.**

Associate Director for Science

Office of the Director, CCR

National Cancer Institute

Building 31, Room 3A11

Bethesda, MD 20892-2440

Tel: 301-496-4345

Fax: 301-496-0775

Email: <benlesmi@mail.nih.gov>

**Ian Blair, Ph.D.**

A.N. Richards Professor of Pharmacology

Center for Cancer Pharmacology

University of Pennsylvania School of Medicine

854 Biomedical Research Building II/III

421 Curie Boulevard

Philadelphia, PA 19104-6160

Tel: 215-573-9880

Fax: 215-573-9889

Email: <ian@spirit.gcrc.upenn.edu>

**Chris Bradfield, Ph.D.**

Professor of Oncology

McArdle Laboratory for Cancer Research

University of Wisconsin Medical School

1400 University Avenue

Madison, WI 53711

Tel: 608-262-2024

Fax: 608-262-2024

Email: <bradfield@oncology.wisc.edu>

**Peter Dedon, M.D., Ph.D.**

Professor

Biological Engineering Division

Massachusetts Institute of Technology

77 Massachusetts Avenue, NE47-277

Cambridge, MA 02139

Tel: 617-253-8017

Fax: 617-324-7554

Email: <pcdedon@mit.edu>

**John Groopman, Ph.D.**

Professor and Chairman

Department of Environmental

Health Sciences

Johns Hopkins University

615 North Wolfe Street, Room W1102

Baltimore, MD 21205

Tel: 410-955-3720

Fax: 410-955-0617

Email: <jgroopma@jhsph.edu>

**Stephen Hecht, Ph.D.**

Wallin Professor of Cancer Prevention

University of Minnesota Cancer Center

420 Delaware Street, S.E.

Mayo Mail Code 806

Minneapolis, MN 55405

Tel: 612-624-7604

Fax: 612-626-5135

Email: <hect002@umn.edu>

**Michael Karin, Ph.D.**

Professor of Pharmacology

Department of Pharmacology

University of California, San Diego

9500 Gilman Drive

La Jolla, CA 92093-0636

Tel: 858-534-1361

Fax: 858-534-8158

Email: <karinoffice@ucsd.edu>

**Larry Marnett, Ph.D.**

Professor, Department of Biochemistry

Director, Vanderbilt Institute of

Chemical Biology

Vanderbilt University School of Medicine

Nashville, TN 37232-0146

Tel: 615-343-7329

Fax: 615-343-7534

Email:

<marnett@toxicology.mc.vanderbilt.edu>

**Leona Samson, Ph.D.**

Professor of Toxicology and Director of the

Center for Environmental Health Sciences

Department of Biological Engineering

Massachusetts Institute of Technology

77 Massachusetts Avenue, 56-235

Cambridge, MA 02139

Tel: 617-258-7813

Fax: 617-253-8099

Email: <lsamson@mit.edu>

**David Schauer, D.V.M., Ph.D.**

Associate Professor

Department of Biological Engineering

Massachusetts Institute of Technology

77 Massachusetts Avenue, 56-787

Cambridge, MA 02139

Tel: 617-253-8113

Fax: 617-258-0225

Email: <schauer@mit.edu>

**Rolf Schulte-Hermann, Ph.D.**

Professor of Toxicology

Institute of Cancer Research

University of Vienna

Borschkegasse 8A

A-1090 Vienna

Austria

Tel: 0043-1-4277-65131

Fax: 0043-1-427-765193

Email:

<rolf.schulte-hermann@meduniwien.ac.at>

**James A. Swenberg, D.V.M., Ph.D.**

Director, Curriculum in Toxicology

University of North Carolina

356 Rosenau Hall, Campus Box 7400

South Columbia Street

Chapel Hill, NC 27599-7400

Tel: 919 966-6139

Fax: 919-966-6123

Email: <james_swenberg@unc.edu>

**Cheryl Walker, Ph.D.**

Professor, Ruth and Walter Sterling\
Professor of Carcinogenesis

University of Texas M.D. Anderson

Cancer Center

1808 Park Road 1C

Smithville, TX 78957

Tel: 512-237-9550

Fax: 512-237-2475

Email: <cwalker@odin.mdacc.tmc.edu>

**CHAIRPERSON:**

**Gerald Wogan, Ph.D.**

Underwood Prescott Professor of Toxicology

Department of Biological Engineering

Massachusetts Institute of Technology

77 Massachusetts Avenue, 26-009

Cambridge, MA 02139

Tel: 617-253-3188

Fax: 617-258-0499

Email: <wogan@mit.edu>

**Epigenetics Think Tank**

**May 19-21, 2004**

**PARTICIPANT ROSTER**

**Timothy Bestor, Ph.D.**

Professor

Department of Genetics and Development

College of Physicians and Surgeons

701 West 168th Street

New York, NY 10032

Tel: 212-305-5331

Fax: 212-740-0992

Email: <thb12@columbia.edu>

**Manel Esteller, M.D., Ph.D.**

Director

Department of Cancer Epigenetics

Spanish National Cancer Centre

Melchor Fernandez Almagro 3

28029 Madrid

Spain

Tel: 34- 91-2246940

Fax: 34-91-2246923

Email: <mesteller@cnio.es>

**Andrew Feinberg, M.D., Ph.D.**

Professor

Epigenetics Unit

Johns Hopkins University

720 Rutland Avenue, Ross 1064

Baltimore, MD 21205

Tel: 410-614-3489

Fax: 410-614-9819

Email: <afeinberg@jhu.edu>

**Stanley Gartler, Ph.D.**

Professor Emeritus

Department of Medicine and Genome

Sciences

University of Washington

J-241 Health Sciences Building

Box 357730

Seattle, WA 98195

Tel: 206-543-1775

Fax: 206-543-1457

Email: <gartler@genetics.washington.edu>

**Michael Goggins, M.D.**

Associate Professor

Department of Pathology, Medicine,

Oncology

Johns Hopkins Medical Institutions

632 Ross Building, 720 Rutland Avenue

Baltimore, MD 21205-2196

Tel: 410-955-3511

Fax: 410-614-0671

Email: <mgoggins@jhmi.edu>

**Shiv Grewal, Ph.D.**

Senior Investigator

Laboratory of Molecular Cell Biology, CCR

National Cancer Institute

Executive Plaza North, Room 6050

6130 Executive Boulevard

Rockville, MD 20852

Tel: 301-594-6389

Fax: 301-435-3697

Email: <grewals@mail.nih.gov>

**Michael Grunstein, Ph.D.**

Professor

Department of Biological Chemistry

David Geffen School of Medicine at UCLA

University of California, Los Angeles

Boyer Hall, Room 302

611 Young Drive East

Los Angeles, CA 90095

Tel: 310-825-0840

Fax: 310-206-9073

Email: <mg@mbi.ucla.edu>

**Steven Henikoff, Ph.D.**

Investigator, Basic Sciences Division

Howard Hughes Medical Institute

Fred Hutchinson Cancer Research Center

1100 Fairview Avenue North

Seattle, WA 98109-1024

Tel: 206-667-4515

Fax: 206-667-5889

Email: <steveh@fhcrc.org>

**Chih-Lin Hsieh, Ph.D.**

Associate Professor

Department of Urology Biochemistry, and

Molecular Biology

University of Southern California

1441 Eastlake Avenue, NOR 5420

Los Angeles, CA 90033

Tel: 323-865-0567

Fax: 323-865-3019

Email: <hsieh_c@ccnt.hsc.usc.edu>

**Randy Jirtle, Ph.D.**

Profesor

Department of Radiation Oncology

Duke University Medical Center

P.O. Box 3433

Durham, NC 27710

Tel: 919-684-2770

Fax: 919-684-5584

Email: <jirtle@radonc.duke.edu>

**Terumi Kohwi-Shigematsu, Ph.D.**

Senior Scientist, Life Sciences Division

Lawrence Berkeley National Laboratory

University of California

1 Cyclotron Road, Mail Box 84-171

Berkeley, CA 94720

Tel: 510-486-4983

Fax: 510-486-4545

Email: <terumiks@lbl.gov>

**Peter Laird, Ph.D.**

Associate Professor

Department of Surgery

University of Southern California

1441 Eastlake Avenue, NOR 6418

Los Angeles, CA 90033

Tel: 323-865-0650

Fax: 323-865-0158

Email: <plaird@usc.edu>

**Kathrin Muegge, M.D.**

Principal Investigator

Laboratory of Molecular

Immunoregulation, SAIC

NCI-Frederick

P.O. Box B, Building 469, Room 243

Frederick, MD 21702-1201

Tel: 301-846-1386

Fax: 301-846-7077

Email: <muegge@ncifcrf.gov>

**Rolf Ohlsson, Ph.D.**

Professor

Department of Developmental Genetics

Uppsala University

Norbyvagen 18A,

Uppsala, S-75236

Sweden

Tel: 46-18-471-2660

Fax: 46-18-4712683

Email: <rolf.ohlsson@ebc.uu.se>

**Benjamin Tycko, M.D., Ph.D.**

Associate Professor of Pathology

Institute for Cancer Genetics

Columbia University

Berrie 412, 1150 St. Nicholas Avenue

New York, NY 10032

Phone: 212-851-5280

Fax: 212-851-5284

Email: <bt12@columbia.edu>

**Patrick Varga-Weisz, Ph.D.**

Chromatin Dynamics Laboratory

Marie Curie Research Institute

The Chart, Oxted

Surrey RH8 0TL

United Kingdom

Phone: 44-1883-722-306

Fax: 44-1883-714-375

Email: <p.varga-weisz@mcri.ac.uk>

**Inflammation and Cancer Think Tank**

**June 2-4, 2004**

**PARTICIPANT ROSTER**

**Raymond N. DuBois, M.D., Ph.D. (Chair)**

Hortense B. Ingram Professor of\
Molecular Oncology

Director of Cancer Prevention

Vanderbilt-Ingram Cancer Center

694 Preston Research Building

2300 Pierce Avenue

Nashville, TN 37232-6838

Tel: 615-343-0527

Fax: 615-936-6865

Email: <raymond.dubois@vanderbilt.edu>

**Jeffrey M. Arbeit, M.D.**

Professor

Department of Surgery and Urology

Washington University School of Medicine

660 South Euclid Avenue, Campus Box 8242

St. Louis, MO 63110

Tel: 314-362-3228

Fax: 314-362-3246

Email: <arbeitj@msnotes.wustl.edu>

**Glenn Dranoff, M.D.**

Associate Professor

Department of Medical Oncology

Dana-Farber Cancer Institute

44 Binney Street, Dana 510E

Boston, MA 02115

Tel: 617-632-5051

Fax: 617-632-5167

Email: <glenn_dranoff@dfci.harvard.edu>

**Isaiah J. Fidler, D.V.M., Ph.D.**

Professor and Department Chairman

Department of Cancer Biology

University of Texas M.D. Anderson\
Cancer Center

1515 Holcombe Boulevard, Unit 173

Houston, TX 77030

Tel: 713-792-8580

Fax: 713-792-8747

Email: <ifidler@mdanderson.org>

**Steven H. Itzkowitz, M.D.**

The Dr. Burrill B. Crohn Professor of Medicine

The Dr. Henry D. Janowitz Division\
of Gastroenterology

Director, GI Fellowship Program

Mount Sinai School of Medicine

GI Division, Box 1069

One Gustave Levy Place

New York, NY 10029

Tel: 212-241-6749

Fax: 212-348-7428

Email: <steven.itzkowitz@msnyuhealth.org>

**Janusz Jankowski, Ph.D.**

Professor

University of Leicester

University Road

Leicester LE1 7RH

United Kingdom

Tel: 0116-252-2522

Fax: 0116-252-2200

Email: <jj29@leicester.ac.uk>

**William G. Nelson, M.D., Ph.D.**

Professor of Oncology, Urology, Pharmacology, Medicine, Pathology, and
Radiation Oncology

Sidney Kimmel Comprehensive Cancer Center

Johns Hopkins Univ. School of Medicine

Bunting-Blaustein Cancer Res. Building,

1650 Orleans Street, Room 151

Baltimore, MD 21231-1000

Tel: (410) 614-3252

Fax: (410) 502-9817

Email: <bnelson@jhmi.edu>

**Richard M. Peek, Jr., M.D.**

Associate Professor

Department of Medicine and Cancer Biology

Vanderbilt University Medical Center

Division of Gastroenterology

C-2104 Medical Center North

1121 21st Avenue South

Nashville, TN 37232

Tel: (615) 322-5200

Fax: (615) 343-6229

Email: <richard.peek@vanderbilt.edu>

**Jeffrey W. Pollard, Ph.D.**

Director, Center for Study of Reproductive\
Biology and Womens Health

Department of Developmental and\
Molecular Biology

Albert Einstein College of Medicine

Chanin Building, Room 607

1300 Morris Park Avenue

Bronx, NY 10461

Tel: 718-430-2090

Fax: 718-430-8972

Email: <pollard@aecom.yu.edu>

**Barrett J. Rollins, M.D., Ph.D.**

Associate Professor of Medicine

Department of Medical Oncology

Dana-Farber Cancer Institute

44 Binney Street

Boston, MA 2115

Tel: 617-632-3896

Fax: 617-632-5998

Email: <barrett_rollins@dfci.harvard.edu>

**Hans Schreiber, M.D., Ph.D.**

Professor

Department of Pathology

University of Chicago

SBRI, Room G-308, MC3008

5841 South Maryland Avenue

Chicago, IL 60637

Tel: 773-702-9204

Fax: 773-702-9224

Email: <hszz@uchicago.edu>

**Robert D. Schreiber, Ph.D.**

Alumni Endowed Professor

Department of Pathology and Immunology

Washington University School of Medicine

660 South Euclid Avenue, Campus Box 8118

St. Louis, MO 63110

Tel: 314-362-8747

Fax: 314-747-4888

Email: <schreiber@immunology.wustl.edu>

**Bonnie F. Sloane, Ph.D.**

Professor and Chair

Department of Pathology

Barbara Ann Karmanos Cancer Institute

Wayne State University School of Medicine

540 East Canfield, Room 6374

Detroit, MI 48201

Tel: 313-577-1580

Fax: 313-577-6739

Email: <bsloane@med.wayne.edu>

**Zena Werb, Ph.D.**

Professor and Vice-Chair

Department of Anatomy

University of California, San Francisco

513 Parnassus Avenue, Box 0452, HSW-1321

San Francisco, CA 94143-0452

Tel: 415-476-4622

Fax: 415-476-4565

Email: <zena@itsa.ucsf.edu>

**Gerald N. Wogan, Ph.D.**

Underwood-Prescott Professor Emeritus

Biological Engineering Division and Department of Chemistry

Massachusetts Institute of Technology

MIT Room 26-009

Cambridge, MA 02139

Tel: 617-253-3188

Fax: 617-258-0499

Email: <wogan@mit.edu>

**Cancer Susceptibility and Resistance Think Tank**

**September 12-14, 2004**

**PARTICIPANT ROSTER**

**Hoda Anton-Culver, Ph.D.**

Professor and Chief, Epidemiology Division

Department of Medicine

University of California, Irvine

224 Irvine Hall

Irvine, CA 92697-7550

Tel: 949-824-7416

Fax: 949-824-4773

Email: <hantoncu@uci.edu>

**Allan Balmain, Ph.D.**

Professor

Biomedical Science Program

Comprehensive Cancer Center

University of California, San Francisco

2340 Sutter Street, Box 0875

San Francisco, CA 94143-0857

Tel: 415-502-4192

Fax: 415-502-0878

Email: <abalmain@cc.ucsf.edu>

**Alan Blatecky, M.S.**

Deputy Director

Renaissance Computing Institute

University of North Carolina at Chapel Hill

3175 Campus Box, Sitterson Hall

Chapel Hill, NC 27599

Tel: 919-962-1871

Fax: 919-962-1799

Email: <blatecky@email.unc.edu>

**Mark Ellisman, Ph.D.**

Professor of Neurosciences and

Bioengineering and Director, Center for Research in Biological Structure

University of California, San Diego

9500 Gilman Drive MC 0608

Basic Science Building, Room 1000

La Jolla, CA 95093-0608

Tel: 858-534-2251

Fax: 858-534-4794

Email: <mark@ncmir.ucsd.edu>

**George Michaels, Ph.D.**

Associate Laboratory Director

Computational and Information Science

Pacific Northwest National Laboratory

P.O. Box 999, Battelle Boulevard

Richland, WA 99352

Tel: 509-372-6464

Fax: 509-375-2668

Email: <george.michaels@pnl.gov>

**Jason Moore, Ph.D.**

Frank Lane Research Scholar in

Computational Genetics

Department of Genetics

Dartmouth College

706 Rubin Building, HB 7937

One Medical Center Drive, DHMC

Lebanon, NH 03756

Tel: 603-653-9939

Fax: 603-653-9900

Email: <jason.h.moore@dartmouth.edu>

**Gilbert Omenn, M.D., Ph.D.**

Professor

Department of Internal Medicine, Human

Genetics and Public Health

University of Michigan

1150 W. Medical Center Drive

A520C, MSRBI

Ann Arbor, MI 48109

Tel: 734-763-7583

Fax: 734-647-8148

Email: <gomenn@umich.edu>

**John Potter, M.D., Ph.D.**

Senior Vice President and Director

Division of Public Health Sciences

Fred Hutchinson Cancer Research Center

1100 Fairview Avenue North, M4-B814

P.O. Box 19024

Seattle, WA 98109-1024

Tel: 206-667-4683

Fax: 206-667-7850

Email: <jpotter@fhcrc.org>

**Nicholas Schork, Ph.D.**

Professor

Department of Psychiatry School of

Medicine

University of California, San Diego

9500 Gilman Drive MC 0608

La Jolla, CA 92093-0603

Tel: 858-822-5788

Fax: 858-822-2113

Email: <nschork@ucsd.edu>

**David Threadgill, Ph.D.**

Associate Professor

Department of Genetics

Medical Biomolecular Research Building

University of North Carolina

Room 434, Campu Box 7264

Chapel Hill, NC 27599

Tel: 919-843-6472

Fax: 919-966-3292

Email: <dwt@med.unc.edu>

**Douglas Wallace, Ph.D.**

Donald Bren Professor of Molecular Medicine/Director, Center for
Molecular and Mitochondrial Medicine and Genetics

University of California, Irvine

Hewitt Hall, Room 2028

Irvine, CA 92697-3940

Tel: 949-824-3490

Fax: 949-824-6388

Email: <dwallace@uci.edu>

**Janet Wiles, Ph.D.**

Professor

Information Technology and Electrical

Engineering Complex and Intelligent

Systems Group

University of Queensland

Brisbane, Australia

QLD 4072

Tel: 617-3365-2902

Fax: 617-3365-4999

Email: <j.wiles@itee.uq.edu.au>

**Tim Wiltshire, Ph.D.**

Department of Genetics

Genomics Institute of the Novartis

Research Foundation

10675 John Jay Hopkins Drive

San Diego, CA 92121

Tel: 858-812-1617

Fax: 858-812-1918

Email: <timw@gnf.org>

**Ming You, M.D., Ph.D.**

Department of Surgery

Alvin J. Siteman Cancer Center

Washington University School of Medicine

660 South Euclid Avenue

Campus Box 8109

St. Louis, MO 63110

Tel: 314-362-9379

Fax: 314-362-9366

Email: <youm@wustl.edu>

**Division of Cancer Biology**

**Scientific Staff**

\* Organizers of the Meetings

**\*Grace Ault, Ph.D.**

Program Director

Tumor Biology and Metastasis Branch

**Donald Blair, Ph.D.**

Program Director

Cancer Cell Biology Branch

**\*Ms. Robin Brown**

Program Specialist

Division of Cancer Biology

**John Cole, Ph.D.**

Program Director

Cancer Etiology Branch

**\*Jennifer Couch, Ph.D.**

Program Director

Structural Biology and Molecular Applications Branch

**Mr. Phillip Daschner**

Program Associate Director

Cancer Etiology Branch

**Ms. Yvonne Duglas-Tabor**

Program Associate Director

Cancer Immunology and Hematology Branch

**\*Dan Gallahan, Ph.D.**

Branch Chief

Structural Biology and Molecular Applications Branch

**Ms. Karen Grotzinger**

Program Associate Director

Cancer Etiology Branch

**\*Jack Gruber, Ph.D.**

Branch Chief

Cancer Etiology Branch

**Ms. Anne Heath**

Program Associate Director

Structural Biology and Molecular Applications Branch

**Ms. Kelly Kim**

Program Associate Director

DNA and Chromosomal Aberrations Branch

**J. Randy Knowlton, Ph.D.**

Program Director

Structural Biology and Molecular Applications Branch

**David Longfellow, Ph.D.**

Senior Coordinator/Carcinogenesis

Cancer Etiology Branch

**\*Carol MacLeod, Ph.D.**

Senior Program Director\
Tumor Biology and Metastasis Branch

**\*Cheryl Marks, Ph.D.**

Associate Director

Division of Cancer Biology

**\*Susan McCarthy, Ph.D.**

Program Director

Cancer Immunology and Hematology Branch

**Judy Mietz, Ph.D.**

Branch Chief

DNA and Chromosomal Aberrations Branch

**\*Suresh Mohla, Ph.D.**

Branch Chief

Tumor Biology and Metastasis Branch

**\*R. Allan Mufson, Ph.D.**

Branch Chief

Cancer Immunology and Hematology Branch

**\*Paul Okano, Ph.D.**

Program Director

DNA and Chromosomal Aberrations Branch

**\*Richard Pelroy, Ph.D.**

Program Director

DNA and Chromosomal Aberrations Branch

**Mary Ellen Perry, Ph.D.**

Program Director

Cancer Cell Biology Branch

**\*Alan Poland, M.D.**

Program Director

Cancer Etiology Branch

**Elizabeth Read-Connole, Ph.D.**

Program Director

Cancer Etiology Branch

**\*Neeraja Sathyamoorthy, Ph.D.**

Program Director,

Tumor Biology and Metastasis Branch

**Ms. Christine Siemon**

Program Associate Director

Tumor Biology and Metastasis Branch

**\*Dinah Singer, Ph.D.**

Division Director

Division of Cancer Biology

**\*John Sogn, Ph.D.**

Deputy Division Director

Division of Cancer Biology

**\*Barbara Spalholz, Ph.D.**

Branch Chief

Cancer Cell Biology Branch

**Ms. Jennifer Strasburger**

Program Associate Director

Cancer Cell Biology Branch

**Bruce Wachholz, Ph.D.**

Sr. Coordinator for International Relations

Division of Cancer Biology

**Mr. Stephen White**

Special Assistant to the Director

Division of Cancer Biology

**May Wong, Ph.D.**

Program Director

Cancer Etiology Branch

**Shen Yang, Ph.D.**

Program Director

Cancer Etiology Branch

**\*Ms. Ellen Zaika**

Program Associate Director

Cancer Etiology Branch

**Acknowledgements**

The Think Tanks in Cancer Biology were only possible because of the more
than one hundred fifty outstanding extramural and intramural scientists
who served willingly and enthusiastically as participants in one or more
of the Think Tanks. The meeting rosters are included to recognize their
contributions. Special thanks are due to the Chairs and Co-Chairs, who
played critical roles in organizing the meetings and summarizing the
recommendations, and to a few individuals who participated in more than
one Think Tank. The overlapping participation contributed a level of
continuity to the discussions.

The Think Tanks also owe much to the participation of the scientific
staff members of the Division of Cancer Biology (DCB), who were involved
in the choice of topics, the planning, the discussions during the
meetings, and the post-meeting retreat that gave rise to this report.
These same individuals will be responsible for implementing many of the
final recommendations. Each Think Tank had one or more principal
internal organizers, and their involvement is recognized on the title
page of each individual report. Several of the Think Tanks, however,
were organized by active committees, and the principal organizers served
as committee chairs. In these cases, the entire committee is not listed
on the relevant report, but an asterisk is placed next to the name of
all DCB staff members who contributed in a major way to the organization
of one or more of the Think Tanks.

Last, but certainly not least, it is a pleasure to acknowledge the lead
administrative role of Ms. Robin Brown in DCB. Robin dealt personally
with all of the logistical details of most of the Think Tanks, in
coordination most of the others with a support contractor, Palladian
Partners. Robin served as Project Officer on the contract. Without her
assistance, the organization and conduct of the Think Tanks would have
been a burden on the entire Division. Instead, the organization was an
enjoyable experience. Finally, it is a pleasure to acknowledge the
efforts of Ms. Ellen Zaika, also of DCB, who played the lead
administrative role for the Cancer Etiology Think Tank.