Immunology and Molecular Oncology in Gynecologic Cancer

KEY TERMS AND DEFINITIONS

Active Immunity. A form of adaptive immunity that is the result of exposure to a foreign
antigen and activation of lymphocytes.
Adaptive Immunity. Also known as specific immunity. The use of extracts derived from
sensitized lymphocytes to transfer "immunologic memory" and induce an antitumor
response.
Adenovirus. An RNA virus that can be used as a vector in gene therapy.
Allele. The form of DNA sequences in a gene on a given chromosomal locus. If the genes
are identical, the individual is homozygous. Different genes result in heterozygosity. One
allele is inherited from the mother and the other from the father.
Amplification. Increase in the number of copies of a DNA sequence in a cell.
Angiogenesis. The formation of new blood vessels. A process in neoplasia whereby
malignant cells release substances that eventually lead to the activation and production of
endothelial cells and the establishment of new capillary blood supply to a growing tumor.
Antibody. Produced by B lymphocytes. Also known as an immunoglobulin. Binds
antigen with high specificity to result in neutralization of antigen, activation of
complement, or leukocyte-dependent destruction of antigen.
Antigen. A molecule that can bind an antibody or T-cell receptor (TCR).
Antigen-Presenting Cell (APC). A cell, often a macrophage or dendritic cell, that digests
the antigen involved in cellular immunity and then displays the foreign antigen as peptide
fragments on its surface in conjunction with major histocompatibility complex (MHC)
molecules to activate T cells. APCs also express molecules to specifically activate T
cells.
Apoptosis. The process of programmed cell death leading to degradation of DNA,
fragmentation of the cell, and phagocytosis by macrophages.
B Lymphocyte. A central mediator of the humoral immune response. B lymphocytes
produce antibodies in response to an antigenic stimulus. They differentiate into plasma
cells, which secrete antibody.
BRCA1, BRCA2. Breast cancer and ovarian susceptibility genes. Thought to function
primarily as DNA repair genes. Mutation of BRCA1 or BRCA2 confers a high lifetime
risk of breast or ovarian cancer.
Cellular Immunity. A form of adaptive immunity in which lymphoid cells, usually CD4+
T cells, activate macrophages or CD8+ cells and directly kill infected cells.
Cluster of Differentiation (CD). Cell surface markers on the surface of T cells that
recognize antibodies and allow for characterization of specific T-cell subunits.
Colony-Stimulating Factors. Cytokines that promote the production of specific progenitor
cells within the bone marrow such as red blood cells, granulocytes, and lymphocytes.
Complement. A component of the immune system consisting of serum and cell surface
proteins activated by antigen-antibody complexes (the classic pathway) or microbial
surfaces (alternative pathway) to generate an inflammatory response and cytotoxicity.
Cytokines. Modulators of the immune and inflammatory system that act as
communicators between members of the immune system.

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Cytotoxic T Lymphocytes. CD8+-expressing cells that kill cells expressing peptides in
conjunction with MHC class I molecules.
Epitope. The portion of an antigen to which an antibody binds, e.g., the portion of an
antigen that binds an MHC molecule that is recognized by a TCR.
Fab. The variable portion of the immunoglobulin molecule that binds to the antigen.
Fc. The "constant" region of the immunoglobulin molecule that is responsible for
biologic activity. It allows for antibody binding to a phagocyte.
Helper T Cells. A T-cell lymphocyte subset that is usually CD4+. Their main effector
function is to activate macrophages in cell-mediated immunity and stimulate B-cell
immunoglobulin production in humoral immunity.
Humoral Immunity. A type of adaptive immunity mediated by antibody production from
B lymphocytes.
Immunoglobulin. Synonymous with antibody. Five basic types are recognized (IgG, IgM,
IgA, IgD, IgE).
Interferon (IFN). A cytokine produced by lymphocytes or fibroblasts in response to viral
infection. There are three types: alpha, beta, and gamma. IFN-γ is produced by T cells
and natural killer (NK) cells and activates macrophages.
Interleukin (IL). A class of cytokines secreted by monocytes, lymphocytes, and
macrophages. They are numerically designated.
Lymphokine. See Cytokine.
Macrophage. A cell derived from monocytes that can act as an APC, phagocytotic cell, or
a producer of cytokines.
Major Histocompatibility Complex. A cluster of genes coded for on chromosome 6 that
include HLA antigens. The gene complex is found in nearly all nucleated cells of the
body and codes for the peptide-binding molecules recognized by T lymphocytes. The
MHC molecules include class I and II MHC molecules. Class I molecules bind peptides
derived from cytosolic proteins and are recognized by CD8+ cells. Class II molecules
bind peptides endocytosed by APCs and are recognized by CD4+ T cells.
Mutation. An alteration of normal genetic composition that leads to a change in the DNA
sequence that is perpetuated in subsequent cell divisions.
Natural Killer (NK) Cell. A type of lymphocyte that produces IFN-γ and functions in the
innate immune system to directly kill cells.
Oncogene. A class of genes that can be mutated, overexpressed, or amplified and is
associated with the development of malignant growth.
Opsonization. A process whereby opsonins, IgG, or complement fragments are attached
to the cell surface of marked cells to target them for phagocytosis.
Overexpression. Increase in the amount of protein product produced by a gene.
Passive Immunity. The transfer of specific antibodies or lymphocytes from one individual
who has responded to and is immune to an antigen to another individual to try to enhance
the immune response.
Plasma cell. Terminally differentiated B lymphocyte that secretes antibody.
Proto-oncogene. A normal gene component in cells that plays a role in physiologic
growth and development. When abnormally activated, they become oncogenes.
Retrovirus. An RNA tumor virus that, when integrated into certain animal cells, leads to
oncogene development. It has an RNA genome and can propagate by reverse
transcription of its RNA into DNA.

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Suppressor T Cell. A differentiated T-cell lymphocyte that functions to suppress B-cell
production and T-cell cytotoxicity.
T Lymphocytes. Lymphocytes that exhibit cell surface antigenicity via the TCR and
mediate cellular immunity within the adaptive immune system. Subsets include CD4+ T
helper cells and CD8+ cytotoxic T lymphocytes.
T-Cell Receptor (TCR). An antigen receptor present on CD4+ and CD8+ T cells that
recognizes peptides complexed with MHC molecules on APCs.
Transfection. The transfer of DNA sequences (genetic material) into a cell.
Translocation. The transfer or exchange of genetic material between two nonhomologous
chromosomes.
Transcription. Process by which messenger RNA is synthesized from a DNA template in
the nucleus, then enters the cytoplasm, and serves as a template for protein synthesis.
Tumor Necrosis Factor (TNF). A cytokine that mediates endotoxic shock and is capable
of inhibiting tumor cell growth. It is produced by activated mononuclear phagocytes and
recruits neutrophils and monocytes to sites of infection.
Tumor-Specific Antigen. Antigen expression restricted to tumor cells and not normal
cells. They can act as targets for immunotherapy.
Tumor Suppressor Gene. A normal genetic component of the cell that controls cell
growth and proliferation. Mutations in the gene can lead to malignancy.

This chapter summarizes general principles of tumor immunology, which is followed by

a description of molecular genetics, signaling pathways, and regulation of angiogenesis
as they apply to gynecologic tumors. These are rapidly expanding fields, and it is not
possible in this chapter to explore them in great detail. However, in the following
sections, some basic immunologic mechanisms are reviewed and the possible application
of this information to gynecologic tumor therapy is explored. This is followed by a
summary of the molecular and genetic changes associated with malignant cellular growth
and a consideration of the application of this knowledge to cancer therapy.

THE IMMUNOLOGIC RESPONSE

The immune system has adapted to fight off foreign intruders such as bacteria and
viruses, but it also plays a role in the surveillance and control of cancer cell growth. The
immune system is divided into that part that is innate, present at birth, and that part that is
adaptive, as a response to infection. Components of the innate immune system include
physical barriers such as epithelial surfaces, macrophages, NK cells, and dendritic cells.
Dendritic cells and macrophages are phagocytic cells that act as APCs. Macrophages also
play an important role in the production of cytokines, which act as growth factors for
other immune cells. Other members of the innate immune system include components of
the complement system and cytokines that regulate these cells. The major components of
the adaptive immune system are the T lymphocytes (T cells) and the B lymphocytes (B
cells). Two types of adaptive immune response exist: cell-mediated immunity and
humoral immunity. Although cell-mediated immunity is mediated by T lymphocytes,
humoral immunity is mediated by antibodies and B lymphocytes.

Innate Immunity

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In contrast to the adaptive immune system that can recognize a variety of foreign
substances including tumor antigens, the innate immune system can only recognize
microbial substances. The main effector cells of the innate immune system include
neutrophils, macrophages, and NK cells. Neutrophils and macrophages are recruited to
sites of infection and inflammation where they phagocytose and destroy microbes.
Receptors on the surface of these effectors, such as the Toll-like receptors recognize
bacterial lipopolysaccharides among other bacterial specific surface molecules to
stimulate the production of cytokines that can recruit additional phagocytic cells to the
site of infection. NK cells are a subset of the lymphocyte population and can directly kill
infected cells. They recognize cells that lack MHC class I molecules such as bacteria.
Recently, Moretta and coworkers reported that NK cells are cytotoxic to tumor cells
likely due to a similar lack of surface MHC class I molecules. NK cells produce IFN-γ,
which activates phagocytic macrophages.

The complement system is also an important component of the innate immune system.
This is a complicated system that consists of a large group of interacting plasma proteins.
Activation by binding to antigen-complexed antibody molecules activates the classic
pathway, whereas the alternative pathway is activated by recognition of microbial surface
structures in the absence of antibody. Activation of these pathways converges in cleavage
of C3 protein into a larger C3b fragment that is deposited on the microbial surface
leading to complement activation and C3a, which acts as a chemoattractant for
neutrophils. Complexing of downstream complement proteins C6, C7, C8, and C9
produces a membrane pore in tagged cells that ultimately results in cell lysis.
Unfortunately, tumor cells seem to be resistant to complement-dependent cytotoxicity.
The innate immune system is intimately linked to the adaptive immune system by cells
such as activated macrophages that enhance T-cell activation and complement fragments
that can activate B cells and antibody production.

Adaptive Immunity

Cellular Immunity: T Cells

Cell-mediated immunity (Fig. 27-1) occurs in response to antigens. APCs such as

macrophages or dendritic cells process the antigen in the context of different MHC
molecules or HLA molecules. The T cells recognize cell surface-associated antigens in
the context of two classes of MHC molecules, class I and II. Specific reactivity of a T-
cell clone depends on antigen tumor-infiltrating by an APC followed by recognition by a
TCR. Unfortunately, not all cancers express class I or II MHC antigens, and, as such, this
can be a mechanism of evading the cellular immune response. The T cells themselves
originate in the bone marrow and differentiate in the thymus. They are found circulating
in the blood or are harbored in the lymph nodes, spleen, or Peyer's patches in the
intestine.

T cells are characterized by their cell surface lymphocyte markers, which are termed CD
groups. T cells, in general, have the CD 2 surface marker, whereas CD 3 is linked to the

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TCR. Th (helper/inducer) cells have a CD 4 surface marker, whereas Ts
(suppressor/cytotoxic) cells have CD 8 surface markers. The CD 4 surface markers are on
T cells that recognize antigens presented with HLA (MHC) class II molecules (Th cell),
and the CD 8 surface markers are on T cells that recognize antigens presented with HLA
(MHC) class I (suppressor/cytotoxic) molecules (Fig. 27-2). In both of these situations,
the CD 2 surface marker is present, and interactions between the TCR and antigen-HLA
complex are stabilized by the CD 3 surface marker.
Within the class of CD4+ helper T cells, there are two helper T cell subsets: Th1 and Th2
cells. Th1 cells produce IFN-α and IL-12, cytokines that induce recruitment of
macrophages and CD8+ cells. Activated CD8+ cells directly lyse infected cells harboring
the antigen determinant or signal B cells to produce antibodies. The latter is mediated
through the production of cytokines. Th2 cells produce IL-4, which induces an antibody
or humoral response from B cells. Recently, Zhang and colleagues reported that the
presence of tumor-infiltrating T cells in ovarian cancer sites has been associated with a
significant improvement in progression-free and overall patient survival.

Humoral Immunity: B Cells and Immunoglobulins

Classes of Immunoglobulins

IgG, the major class of immunoglobulin, comprises approximately 70% of total serum
immunoglobulin and is the predominant form of antibody. Only IgG crosses the placenta
and is important for the transfer of passive immunity to the newborn.

IgA comprises approximately 20% of total serum immunoglobulin. It is found in saliva,

milk colostrums, and genito-urinary secretions.

IgM represents approximately 10% of total serum immunoglobulin. It is the predominant

antibody produced early in the humoral immune responses and is the most efficient in
activating complement.

IgD accounts for less than 1% of total serum immunoglobulin and is found on the surface
of B cells. The precise role of IgD is not understood but presumably it plays a role in B-
lymphocyte activation.

IgE is associated with hypersensitivity and also infection with parasites.

In humans, B cells are derived from hematopoietic stem cells and aggregate in lymph
nodes, the spleen, or the GI tract. Antibodies are synthesized from B lymphocytes in
response to activated CD8+ cells or Th2 cells. In turn, the B lymphocytes differentiate
into plasma cells that secrete large quantities of antibody (immunoglobulin) in response
to the antigenic stimulant. In contrast to the immune response initiated by T cells where
the antigen must be processed, B cells recognize antigens in an unprocessed state. Each B
cell is programmed to secrete a specific type of antibody, and it is estimated that more
than 107 different antibodies are capable of being produced in response to the presence of

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foreign antigens. All antibodies have the same basic structural characteristics but exhibit
extensive variability in the portion of the structure that specifically binds antigen. The
basic immunoglobulin structure consists of two identical heavy chains and two identical
light chains. Each pair is connected by a disulfide bond. Both heavy and light chains have
a variable region (V) at the amino terminal and a constant region (C) at the carboxy
terminal (Fig. 27-3). The V region participates in antigen recognition and confers
specificity, and the C region mediates effector function by allowing the antibody to bind
to a phagocyte. Five types of related immunoglobulin molecules are recognized (IgG,
IgM, IgA, IgD, IgE [see the following box]) that serve different effector functions. IgM
and IgD production occurs early in the antibody response. The membrane-bound form of
IgM and IgD binds antigen and activates naive B cells leading to B-cell proliferation and
clonal selection. IgM is also involved in activation of the classic pathway of the
complement system. IgG production occurs later in the antibody response and is of higher
specificity for particular antigens. IgG is responsible for neonatal immunity in the
transfer of maternal antibodies across the placenta and gut. Its main effector functions
include opsonization of antigen for phagocytosis by macrophages and neutrophils as well
as activation of the classic pathway of the complement system. NK cells and other
leukocytes can bind to IgG- and IgE-coated cells to facilitate antibody-dependent
cytotoxicity. Finally, IgA is responsible for mucosal immunity, whereas IgE mediates
hypersensitivity reactions.

CYTOKINES

Cytokines, proteins secreted by immune cells, mediate other immune cell functions.
During the activation phase of the immune response system, cytokines stimulate the
growth and differentiation of lymphocytes, whereas in the effector phase of the immune
response, they activate other effector cells to help eliminate antigens and microbes. The
major classes of cytokines include those that regulate innate immunity, those that regulate
adaptive immunity, and, finally, those that stimulate hematopoiesis.

Cytokines That Mediate Innate Immunity

Tumor Necrosis Factor

TNF mediates the acute inflammatory response to bacterial infections and microbes. It is
secreted principally by macrophages, T cells, NK cells, and mast cells. TNF is produced
in response to lipopolysaccharide production by bacteria. IFN-γ, which is produced by T
cells and NK cells, can augment TNF production by macrophages. The primary action of
TNF is to recruit neutrophils and monocytes to sites of infection to facilitate eradication
of bacteria. One complication of sepsis, largely as a result of TNF production, is septic
shock, which results in intravascular collapse and disseminated intravascular coagulation
(DIC).

Interleukins

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ILs are potent cytokines that are produced by some leukocytes to affect other leukocytes.
They are designated numerically. IL-1 is released in response to cell damage by
macrophages, endothelial cells, and some epithelial cells. Many of its actions are similar
to those of TNF. However, it lacks the ability to produce the septic shock symptoms that
TNF can. Other important ILs include IL-12, IL-10, IL-6, IL-15, and IL-18. All are
produced by macrophages. IL-12 plays a key role in the transition between cell-mediated
immunity and adaptive immunity. IL-12 stimulates NK cells and T cells to produce IFN-
γ, which activates macrophages to kill phagocytosed foreign substances including
microbes. It also increases cytolytic activity by stimulating CD8+ cells. The other ILs
stimulate NK- and T-cell activation and proliferation as well as IFN-γ synthesis. IFNs
protect against viral infection; increase expression of class I MHC molecules, which
increases cytotoxic T lymphocyte-mediated cytotoxicity; and stimulate production of Th
cells. Marth and colleagues have found that high levels of IFN-γ expression in ovarian
cancer specimens are associated with longer survival in patients.

Cytokines That Mediate Adaptive Immunity

IL-2, IL-4, IL-5, and IL-13 are all involved in regulation of adaptive immunity in
addition to IFN-γ and transforming growth factor β. After T cells recognize antigen, the T
cells produce IL-2, which leads to clonal expansion of activated T cells and additional
production of cytokines such as IFN-γ and IL-4. IL-2 also affects B cells by acting as a
growth factor and stimulus for antibody synthesis. IL-4 plays a key role in stimulating
IgE production from B cells and, most importantly, stimulates the development of Th2
cells from naive T cells. IFN-γ, as mentioned previously, can stimulate class I and II
MHC production in addition to stimulating Th1 differentiation, activating neutrophils,
and promoting cytolytic activity of NK cells. Transforming growth factor β, on the other
hand, inhibits proliferation and differentiation of T cells.

Cytokines That Mediate Hematopoiesis

Colony-Stimulating Factors

IL-3 is a multilineage colony-stimulating factor that allows for differentiation of cells into
all known cell types. Granulocyte colony-stimulating factor, a cytokine, is produced by
macrophages, fibroblasts, and endothelial cells. It promotes mobilization of neutrophils
from the bone marrow. Granulocyte-macrophage colony-stimulating factor is produced
by T cells, macrophages, endothelial cells, and fibroblasts. It stimulates maturation of
bone marrow cells into dendritic cells and monocytes. Both granulocyte colony-
stimulating factor and granulocyte-macrophage colony-stimulating factor are used in
patients undergoing chemotherapy and bone marrow transplant.

TUMOR-CELL KILLING AND IMMUNOTHERAPY

Immunotherapy has been developed to recognize and destroy tumor cells. Three major
classes of immunotherapy exist: immune modulation, passive therapy, and active therapy.
Immune-modulation relies on nonspecific means such as the administration of IL-2,

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IFNs, or bacille Calmette-Guérin to elicit an immune response. Passive therapy transfers
components of the acquired immune system to the recipient with cancer. Examples
include the use of monoclonal antibodies directed toward tumor-specific antigens. Active
therapy makes use of the patient's own immune system to elicit a response. Vaccines
making use of peptides, proteins, DNA, or RNA would fall into this category.

Immune modulation has been used in ovarian cancer in the form of adjuvant IFN
treatment after surgery and as consolidation therapy after surgery and standard
chemotherapy. One randomized prospective trial in patients with advanced-stage ovarian
cancer randomized patients to cisplatin and cyclophosphamide chemotherapy versus the
same regimen with intraperitoneal IFN-γ. The study of Windbichler and coworkers
showed an improvement in both progression-free survival and 3-year survival in the IFN
arm with manageable toxicity. In contrast, prospective studies reported by Alberts and
coworkers and by Hal and associates showed that using IFN-α as consolidation therapy
after surgery and standard chemotherapy did not improve progression-free or overall
survival, but these studies were hampered by patient numbers insufficient to detect a
difference in outcome.

Tumor cells are known to have specific tumor-associated antigens or receptors on their
surface that may distinguish them from normal cells. One example of successful passive
therapy is the use of monoclonal antibodies directed toward overexpressed growth factor
receptors such as Her-2/neu (erb-B2) or epidermal growth factor receptor (EGFR). In
studies reported by Vogel and coworkers and by Slamon, Leyland-Jones, and associates,
the humanized monoclonal antibody trastuzumab (Herceptin) has been used very
successfully in metastatic breast cancer where 20% to 30% of patient tumors overexpress
Her-2/neu. In contrast, in a study by Bookman and associates of ovarian cancer, treatment
with trastuzumab has met with limited success because only 10% of patients overexpress
Her-2/neu. Bevacizumab (Avastin), a monoclonal antibody directed against vascular
endothelial growth factor (VEGF), is currently approved by the U.S. Food and Drug
Administration for frontline treatment with chemotherapy for metastatic colorectal cancer
and has under-gone phase II testing in ovarian cancer, with encouraging results reported
by Burger and coworkers.

One promising immunotherapy that has come out recently has been the human
papillomavirus (HPV) vaccine for the prevention of cervical dysplasia and cervical
cancer. Koutsky and colleagues reported on the use of an HPV 16 virus-like particle
vaccine in 2392 women who were negative for HPV 16 DNA and antibodies at
enrollment. There was a significant reduction in the number of women with persistent
HPV 16 infection in the vaccine group: 0 per 100 woman-years at risk versus 3.8 per 100
woman-years at risk in the placebo group (P < .001). There were no cases of cervical
dysplasia in the vaccine group as opposed to nine in the placebo group. A study
conducted by Villa and coworkers showed that use of a quadrivalent vaccine against
HPV types 6, 11, 16, and 18 decreases the incidence of persistent infection or dysplasia
by 90% (P < .0001). The use of HPV vaccine therapy is efficacious and will have
tremendous implications for the prevention of cervical cancer.

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MOLECULAR ONCOLOGY

A cancer may arise as a result of acquired mutations or through inheritance of a mutated

gene followed by acquisition of an acquired mutation in the other allele. The former is
known as a sporadic cancer, and the latter is known as a hereditary cancer. Genetic
alterations occur in three major categories of genes: oncogenes, tumor suppressor genes,
and DNA mismatch repair genes. Knowledge of how these genes function is a rapidly
expanding field and well beyond the scope of this text, but a general overview is
provided.

The term oncogene has been applied to one set of genes that, when altered, is associated
with the development of a malignant cell. Most studies describing oncogenes have been
done in animals such as chickens, mice, and rats. Many of the oncogenes described in the
literature probably do not play a role in human disease. Functionally, oncogenes are
involved in cell proliferation, signal transduction, and transcriptional alteration.
Mechanisms of alteration in oncogene function include gene amplification (increase in
numbers of copies of genes in the cell), translocation, or overexpression. The term
overexpression refers to excessive and abnormal protein production.

Oncogenes

Several classes of oncogenes exist including the peptide growth factors, cytoplasmic
factors, and nuclear factors. Representative examples are described in the following
sections (Table 27-1).

Peptide Growth Factors

Epidermal Growth Factor Receptor Family

The family of erb-B receptors has been reviewed recently by Britten and includes the
EGFR (erb-B1), Her-2 (erb-B2), erb-B3, and erb-B4. All erb-B receptors share the same
structure including an extracellular domain that binds ligand, a transmembrane domain,
and an intracellular tyrosine kinase domain. EGFR, erb-B3, and erb-B4 can bind their
respective receptors and dimerize with one another. Her-2 does not have an identifiable
ligand and dimerizes with the other receptors in response to ligand binding. A cascade of
phosphorylation events then occurs that ultimately results in cellular processes such as
cell growth, proliferation, and differentiation. Although the erb-B receptors share
activation of some signaling pathways, there are certain pathways that are activated
preferentially by specific receptors. For instance, activation of the erb-B3 receptor
preferentially activates the phosphatidylinositol 3' kinase pathway discussed later. A
number of different mechanisms exist that are associated with malignant transformation.
Activating mutations have been found in the tyrosine kinase domain of EGFR in lung
cancer by Lynch and colleagues and by Paez and associates. This finding has led to the
development of a number of EGFR inhibitors that include monoclonal antibodies directed
at the ligand binding domain and tyrosine kinase inhibitors directed at the catalytic
domain. Gene amplification is also a well-characterized mechanism of EGFR family
alteration. Slamon, Godolphin, and coworkers have reported that Her-2/neu

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amplification, for instance, is found in 20% to 30% of breast cancers and 10% of ovarian
cancers. According to a study by Vogel and coworkers, trastuzumab, a humanized
monoclonal antibody to HER-2/neu, produces a response rate of 34% in women with
HER-2/neu-positive breast cancer as measured by fluorescent in situ hybridization. In
addition to combining trastuzumab with chemotherapeutic agents such as carboplatin,
ongoing studies are also looking at combining trastuzumab with EGFR tyrosine kinase
inhibitors in the hope that multiple downstream signaling pathways necessary for cancer
cell proliferation will be blocked.

Vascular Endothelial Growth Factor and Angiogenesis

The process of angiogenesis involves the development of new vasculature to bring

nutrients and necessary substrates to tumors. One key modulator of this process is VEGF
and its receptors, as reviewed by Ferrara and associates. Tumors frequently express
growth factors such as epidermal growth factor, transforming growth factors α and β and
platelet-derived growth factor that up-regulate VEGF. VEGF, in turn, acts as a potent
mitogen for endothelial cells. Strategies to target VEGF have included the use of a
humanized monoclonal antibody (bevacizumab) directed against VEGF. Currently, this
treatment is approved by the U.S. Food and Drug Administration for use in colorectal
cancer where, in a study reported by Hurwitz and coworkers, it yielded a 30% increase in
median survival in conjunction with chemotherapy when compared with chemotherapy
alone. Bevacizumab has also shown promise in phase II studies as a single agent in
ovarian cancer and will undergo phase III testing as frontline therapy in newly diagnosed,
advanced stage ovarian cancer patients in conjunction with standard paclitaxel and
carboplatin chemotherapy.

Cytoplasmic Factors

Phosphatidylinositol 3' Kinase Pathway

As described by Hennessy and associates, the phosphatidylinositol 3' kinases (PI3Ks) are
lipid kinases that control second messengers governing cellular proliferation, survival,
motility, and morphology. With growth factor stimulation, p110α, the catalytic subunit of
PI3K, phosphorylates a series of proteins, among them AKT, a serine threonine kinase
with multiple isoforms. AKT activates several downstream targets, one of which is
mTOR (mammalian target of rapamycin), a regulator of cellular proliferation and
translation. PTEN (phosphatase and tensin homologue) is the regulatory counterpart to
PI3K and a tumor suppressor gene. It dephosphorylates proteins phosphorylated by PI3K.
Aberrant PI3K signaling in cancer has been attributed to alterations in a number of
members of this pathway. Mutations in p110α were first described in 2004 by Samuels
and associates and may lead to gain of function activity. Levine and colleagues have
found that the PIK3CA gene, which encodes for the p110α catalytic subunit of PI3K, has
missense somatic mutations in 12% of ovarian cancers and 18% of breast cancers. Other
mechanisms of PI3K signaling dysfunction include loss of PTEN function by mutation or
transcriptional down-regulation or gain of AKT function by amplification,
overexpression, or increased phosphorylation, as reported by Bellacosa and coworkers.

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Somatic mutations in PTEN have been described in endometrial and ovarian cancers,
whereas AKT2 amplification has been identified in breast and ovarian cancers. As
reported by Adjei and Hidalfo, current drugs under study that target this pathway include
PI3K and AKT inhibitors as well as mTOR inhibitors such as rapamycin and the
rapamycin analogue CCI-779.

Another class of cytoplasmic factors that is involved in growth stimulation is the G

protein class of RAS genes. Adjei has reported that the protein products of the RAS genes
regulate a number of processes including cellular differentiation and cytoskeletal
organization. This class of genes plays a central role in the regulatory pathway that
controls signal transduction from the cell surface to the nucleus. The Ras proteins localize
to the inner surface of the cell membrane and, in response to activation of cell surface
receptor tyrosine kinases, activate pathways such as the mitogen-activated protein kinase
signaling pathway, which, in turn, activates the production of nuclear transcription factors
such as c-myc, c-jun, and c-fos. In turn, as described by Neves and colleagues and by
Adjei and Hidalgo, these transcription factors modulate a number of proliferation genes.
According to a review article by Bos, oncogenic Ras proteins have been found in 30% of
human cancers. Current therapeutic approaches that target the oncogenic Ras signaling
pathway include the use of antisense oligonucleotides, producing viruses to specifically
kill Ras-transformed cells, and inhibition of downstream Ras effector pathways.

Tumor Suppressor Genes

Tumor suppressor genes restrain cellular proliferation. In this capacity, they serve vital
functions in normal cell regulation. The retinoblastoma gene (Rb) was the first tumor
suppressor gene to be characterized and is described in a 1986 study by Friend and
colleagues. Baretk and coworkers have shown that Rb encodes a nuclear protein that
regulates G1 phase cell cycle arrest. In the inherited form of retinoblastoma, two gene
copies are inherited. Affected individuals inherit one defective copy of the Rb gene, but
because there is a normal intact copy, a malignancy does not develop until the second
copy of the gene is inactivated, presumably as a result of mutation. Thus, there is a "two-
hit" theory of action of the tumor suppressor gene, with the first "hit" being present in the
inherited abnormal gene and the second "hit" the consequence of somatic mutations that
occur later, manifesting in cancer.

The most widely known tumor suppressor gene in gynecologic oncology is the p53 gene,
which is located on the short arm of human chromosome 17, as described by Finlay and
associates. p53 functions as a transcription factor and regulator of the cell cycle and
apoptosis. One key function of normal p53 is to bind to transcriptional regulatory
elements in DNA. As gatekeeper of the genome, in response to DNA damage, p53
activates genes regulating apoptosis (Bax, Fas, bcl-2). Hollstein and coworkers have
shown that the most common mutations in p53 are missense mutations within exons 5 to
8. The resultant mutant proteins can no longer bind to DNA yet can complex with and
inactivate any normal p53 in the cell. Cells expressing mutant p53 do not undergo cell
cycle arrest at the typical G1-S checkpoint before DNA replication nor do they undergo
apoptosis.

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Tumor suppressor genes play an active role in the modulation of gynecologic
malignancy. For instance, the role of Rb and p53 in cervical carcinogenesis attributable to
high-risk HPV types has recently been elucidated. The E6 oncoprotein of HPV types 16
and 18 has been shown by Werness and colleagues and by Scheffner and associates to
bind to p53 and target it for degradation. The E7 oncoprotein of HPV type 16 associates
with the Rb gene product in infected cells to up-regulate proliferation.

Although p53 mutations are one of the most common in human cancer, attempts to target
and modulate this tumor suppressor pathway have met with disappointing results. Use of
an adenovirus to replace defective p53 in recurrent ovarian cancer has not resulted in any
clinically significant benefit to patients in two studies published by Buller and associates
in 2002.

BRCA1 and BRCA2

Approximately 5% to 10% of breast and ovarian cancers have a hereditary basis, as

shown in studies published by Rosen and colleagues and by Risch and coworkers.
According to Frank and colleagues, the vast majority are attributable to the inheritance of
mutations in BRCA1 and BRCA2. In 1990, Hall and associates performed genetic studies
that linked familial breast cancer risk to chromosome 17q21. In 1994, BRCA1, located on
chromosome 17, was identified by positional cloning done by Miki and associates.
Because only 45% of familial breast cancers could be linked to BRCA1, Wooster and
coworkers searched for a second breast cancer susceptibility gene, now known as
BRCA2, which was localized to chromosome 13q12.3 and reported their findings in 1994
and 1995. The pattern of inheritance is autosomal dominant in nearly all families studied.
Approximately 0.5% to 0.6% of U.S. women are carriers of an altered BRCA1 or
BRCA2 gene; however, certain groups such as Ashkenazi Jewish women are at
particularly high risk with more than 1% carrying a mutated gene. It is estimated that for
carriers of BRCA1 mutations, breast cancer will develop by age 70 in 36% to 85% of
women and ovarian cancer in 40% to 60%. Cass, Holscheider, and colleagues have
reported that development of fallopian tube carcinoma is also a part of the BRCA
phenotype. An analysis conducted by Antoniou concluded that the lifetime risk of
development of ovarian cancer with BRCA2 is far lower, approximately 10%.
Inheritance of mutated BRCA2, however, is associated with other cancers including male
breast cancer and pancreatic, urinary tract, and biliary tract cancers. Unfortunately, the
women who develop these hereditary forms of cancer develop breast and ovarian cancers
at a younger age than is typically seen with sporadic cancers. Personal characteristics
associated with an increased likelihood of a BRCA1 or BRCA2 mutation include breast
cancer diagnosed at an early age (younger than age 50), bilateral breast cancer, and/or a
history of both breast and ovarian cancers. Family history characteristics associated with
an increased likelihood of carrying a BRCA1 or BRCA2 mutation include multiple cases
of breast cancer in the family, both breast and ovarian cancers in the family, male breast
cancer, and one or more family members with two primary cancers.

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Sowter and Ashworth reviewed BRCA1 and BRCA2 as tumor suppressor genes that
encode for large proteins, 1863 amino acids and 3418 amino acids, respectively.
Inheritance of a mutated allele followed by inactivation of the normal allele allows for
tumorigenesis. Both BRCA1 and BRCA2 code for proteins that play a role in repair of
DNA strand breaks. There are more than 200 mutations that have been identified in
BRCA1; the vast majority of these mutations result in truncated proteins. BRCA1 has
been found by Scully and coworkers to colocalize in vivo and associate in vitro with
RAD51, a protein known to repair double-stranded DNA breaks. In addition to
interacting directly with RAD51, BRCA2 has also been identified as a FANCD1 gene, a
member of the Fanconi anemia complex of proteins. Cells with deficient BRCA1 or
BRCA2 are incapable of repairing DNA strand breaks, which results in genetic
instability. Why mutations in BRCA1 particularly predispose to breast and ovarian
cancers is unclear. BRCA1 has been shown by Ma and associates to interact with the
estrogen receptor α directly, implicating it as a regulator of preventing estrogen-
independent proliferation. BRCA2 contains a region known as the ovarian cancer cluster
region (nucleotides 4075-6503 in exon 11) where mutations in this region have been
reported by Thompson and the Breast Cancer Linkage Consortium to be associated with
increased ovarian cancer and decreased breast cancer risk. For BRCA1, Gayther and
coworkers have reported that mutations in the C terminal end, required for DNA repair
and transcriptional activation, predispose to breast cancer, whereas mutations in the N
terminal end are associated with development of ovarian cancer. The outcome of patients
with BRCA1 or BRCA2 mutations who develop ovarian cancer has been somewhat
controversial. Recent studies by Boyd and colleagues, Ben David and associates, and
Cass, Baldwin, and coworkers have demonstrated that these women survive longer than
their sporadic cancer counterparts. It has been suggested by Bhattacharyya and colleagues
that this longer survival may be the result of improved response to platinum-based
chemotherapy due to inability to repair chemotherapy-induced DNA damage.

DNA Mismatch Repair Genes

The role of the DNA mismatch repair gene system is to recognize and fix errors in the
DNA helix resulting from incorrect pairings of nucleotides. In addition, the system
repairs single-stranded DNA loops that occur in the helix when an extra nucleotide has
been inserted or deleted in one of the DNA strands. During the repair process, Markowitz
suggests, the system digests the individual bases and resynthesizes the correct sequences.
The most common germline mutations in DNA mismatch repair genes are acquired in an
autosomal-dominant fashion and occur on chromosomes 2 and 3 in the genes MSH2,
MLH1, MSH6, and PMS2, according to a study reported by Fishel and coworkers. More
than 400 mutations in the mismatch repair genes have been identified; Peltomaki reports
that the majority (50%) occur in MLH1, 40% in MSH2, 10% in MSH6, and less than 5%
in PMS2. Patients who acquire these mutations develop chromosomal aberrations and an
accumulation of abnormal cells resulting in hereditary nonpolyposis colon cancer
syndrome. In this syndrome, patients inherit a mutated DNA mismatch repair gene
followed by subsequent loss of the wild-type allele. The clinical criteria for diagnosis of
this syndrome are known as the Amsterdam criteria and consist of three individuals
diagnosed with colon cancer. As described by Vasen and colleagues, the three individuals

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must represent two different generations, one member must be a first-degree relative of
the other two, and one of the three individuals must have developed colon cancer before
the age of 50. The colon cancers are frequently found in the proximal, as opposed to the
distal, colon. In addition to colorectal cancer, cancers of the ovary and endometrium, GI
tract, upper urologic tract, and sebaceous glands (Muir-Torre syndrome) are also
frequently found. Endometrial cancer is the second most common cancer found in this
cohort of patients. The lifetime risk of developing ovarian cancer in patients with
hereditary nonpolyposis colon cancer is 5% to 10%, whereas the risk of developing
endometrial cancer is 40% to 60%.

KEY POINTS

The immune system consists of the innate and adaptive immune systems. The innate
system is present at birth and consists of natural barriers, NK cells, macrophages, and the
complement system. The adaptive immune system adapts to infection or foreign invasion
and consists of the T cells and B cells.
The cellular immune response occurs as a result of T lymphocytes reacting via a surface
TCR that processes antigens presented to it by an APC in conjunction with HLA (MHC)
molecules.
T-cell activation can result in activation of helper/inducer (Th) cells, cytotoxic/suppressor
(Ts) cells, or cytokine production.
Helper/inducer (Th) cells recruit macrophages and cytotoxic/suppressor cells.
Cytotoxic T cells have the ability to lyse infected cells or signal B cells to produce
antibody.
Humoral immunity results from antigen stimulation of a B lymphocyte, which
differentiates into a plasma cell and secretes antibody (immunoglobulin).
There are five types of immunoglobulins (IgG, IgM, IgD, IgA, and IgE). The
immunoglobulin molecule consists of a fixed region (Fc), which carries the biologic
activity, and a variable region (Fab), which reacts to a specific antigen.
The complement cascade provides a basis for the inflammatory response and can also
mediate cytotoxicity.
T cells are characterized by cell surface markers, which are termed clusters of
differentiation (CD), and are numbered CD1 to CD80.
Cytokines (lymphokines) are regulatory substances of the immune system produced as a
result of T-cell activation, cell damage by a virus, or other cells, such as macrophages and
monocytes, involved in the immune response.
IFNs are a group of cytokines produced in response to viral infections. They also have
antiproliferative effects and can enhance the antitumor immune response as well as
interact with other cytokines and chemotherapeutic agents.
Colony-stimulating factors are a group of cytokines that can stimulate various parts of the
bone marrow and can help to overcome the suppressive effects of chemotherapy.
Immune modulation is a nonspecific form of immunotherapy that consists of
administration of IL-2, IFNs, or bacille Calmette-Guérin to elicit an immune response.
Passive therapy transfers components of the acquired immune system to the recipient
with cancer, e.g., monoclonal antibodies directed toward tumor-specific antigens.

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Active immunotherapy uses a patient's own immune system for protection against
infection, e.g., vaccines.
There are three types of genes associated with malignant development: oncogenes, tumor
suppressor genes, and DNA mismatch repair genes.
Proto-oncogenes are protein sequences of oncogenes that occur in normal cells and
regulate physiologic growth and development. When activated, they can lead to
malignant change.
Malignant change is seen with point mutations, chromosomal aberration, gene
amplification (increase in number of copies), or chromosomal translocation.
Overexpression is excessive protein production in the cytoplasm, a result of gene
alterations that can lead to abnormal cell growth.
Ras oncogenes are part of a group of signal transducer oncogenes that relay messages
from the membrane to the cell interior. They are activated generally by point mutations.
Growth factor genes include C-erb-B2 (Her-2/neu), which can be overexpressed and act
as a tumor-specific target for monoclonal antibody therapy.
Nuclear oncogenes include myc and fos and can activate other genes as well as stimulate
DNA replication.
Tumor suppressor genes restrain cell growth. They have two copies, and, in general,
alteration of both copies leads to a mutant expression, which allows malignant growth to
occur.
Rb (retinoblastoma) and p53 are two widely studied tumor suppressor genes.
Mutant p53 prevents normal p53 from interacting with DNA, which prevents cellular
growth inhibition.
BRCA1 and BRCA2 mutations confer a high lifetime risk of breast and/or ovarian
cancer. Mutation screening may be appropriate for women with family histories
suggesting a hereditary predisposition to breast or ovarian cancer.
DNA mismatch repair genes act by recognizing and fixing errors in the DNA helix
resulting from incorrect pairings of nucleotides. They prevent accumulation of
genetically damaged material in the cell.
Angiogenesis is the formation of new capillary blood vessels that can bring nutrients and
necessary substrates to tumors.

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