CHAPTER 6
Molecular Biology and Genetics
KEY POINTS
1 The regulation and maintenance of normal tissue requires a balance between cell
proliferation and programmed cell death, or apoptosis.
2 Among the genes that participate in the control of cell growth and function, protooncogenes and tumor suppressor genes are particularly important.
3 Growth factors trigger intracellular biochemical signals by binding to cell membrane
receptors. In general, these membrane-bound receptors are protein kinases that
convert an extracellular signal into an intracellular signal. Many of the proteins that
participate in the intracellular signal transduction system are encoded by protooncogenes that are divided into subgroups based on their cellular location or
enzymatic function.
4 Oncogenes comprise a family of genes that result from gain of function mutations of
their normal counterparts, proto-oncogenes. The normal function of proto-oncogenes
is to stimulate proliferation in a controlled context. Activation of oncogenes can lead
to stimulation of cell proliferation and development of a malignant phenotype.
5 Tumor suppressor genes are involved in the development of most cancers and are
232usually inactivated in a two-step process in which both copies of the tumor
suppressor gene are mutated or inactivated by epigenetic mechanisms like
methylation. The most commonly mutated tumor suppressor gene in human cancers
is p53.
2 T lymphocytes have a central role in the generation of immune responses by acting as
helper cells in both humoral and cellular immune responses and by acting as effector
cells in cellular responses. T cells can be distinguished from other types of
lymphocytes by their cell surface phenotype, based on the pattern of expression of
various molecules, and by differences in their biologic functions.
2 There are two major subsets of mature T cells that are phenotypically and
functionally distinct: T-helper/inducer cells, which express the CD4 cell surface
marker, and the T-suppressor/cytotoxic cells, which express the CD8 marker. TH1
and TH2 are two helper T-cell subpopulations that control the nature of an immune
response by secreting a characteristic and mutually antagonistic set of cytokines:
Clones of TH1 produce interleukin-2 (IL-2) and interferon-γ (IFN-γ), whereas TH2
clones produce IL-4, IL-5, IL-6, and IL-10.
Advances in molecular biology and genetics have improved our understanding of
basic biologic concepts and disease development. The knowledge acquired with
the completion of the human genome project, the data available through The
Cancer Genome Atlas (TCGA), the development of novel diagnostic modalities,
such as the microarray technology for the analysis of DNA and proteins, and the
emergence of treatment strategies that target specific disease mechanisms all have
an increasing impact on the specialty of obstetrics and gynecology.
Normal cells are characterized by discrete metabolic, biochemical, and
physiologic mechanisms. Specific cell types differ with respect to their mainly
genetically determined responses to external influences (Fig. 6-1). An external
stimulus is converted to an intracellular signal, for example, via a cell membrane
receptor. The intracellular signal is transferred to the nucleus and generates
certain genetic responses that lead to changes in cellular function, differentiation,
and proliferation. Although specific cell types and tissues exhibit unique
functions and responses, many basic aspects of cell biology and genetics are
common to all eukaryotic cells.
CELL CYCLE
Normal Cell Cycle
Adult eukaryotic cells possess a well-balanced system of continuous
production of DNA (transcription) and proteins (translation). Proteins are
constantly degraded and replaced depending on the specific cellular
233requirements. Cells proceed through a sequence of phases called the cell
cycle, during which the DNA is distributed to two daughter cells (mitosis)
and subsequently duplicated (synthesis phase). This process is controlled at key
checkpoints that monitor the status of cells, for example, the amount of DNA
present in each cell. The cell cycle is regulated by a small number of
heterodimeric protein kinases that consist of a regulatory subunit (cyclin) and a
catalytic subunit (cyclin-dependent kinase). Association of a cyclin with a cyclindependent kinase (CdkC) determines which proteins will be phosphorylated at a
specific point during the cell cycle.
The cell cycle is divided into four major phases: M phase (mitosis), G1
phase (period between mitosis and initiation of DNA replication), S phase
(DNA synthesis), and G2 phase (period between completion of DNA synthesis
and mitosis) (Fig. 6-2). Post-mitotic cells can “exit” the cell cycle into the socalled G0 phase and remain for days, weeks, or even a lifetime without
further proliferation. The duration of the cell cycle may be highly variable,
although most human cells complete the cell cycle within approximately 24
hours. During a typical cell cycle, mitosis lasts about 30 to 60 minutes, the G1
phase 7 to 10 hours, S phase 10 hours, and G2 phase 5 hours. With respect to the
cell cycle, there are three subpopulations of cells:
FIGURE 6-1 External stimuli affect the cell, which has a specific coordinated response.
234FIGURE 6-2 The cell cycle.
1. Terminally differentiated cells cannot reenter the cell cycle.
2. Quiescent (G0) cells can enter the cell cycle if appropriately stimulated.
3. Dividing cells are currently in the cell cycle.
Red blood cells, striated muscle cells, uterine smooth muscle cells, and nerve
cells are terminally differentiated. Other cells, such as fibroblasts, exit from the
G1 phase into the G0 phase and are considered to be out of the cell cycle. These
235cells enter the cell cycle following exposure to specific stimuli, such as growth
factors and steroid hormones. Dividing cells are found in the gastrointestinal tract,
the skin, and the cervix.
G1 Phase
In response to specific external stimuli, cells enter the cell cycle by moving
from the G0 phase into the G1 phase. The processes during G1 phase lead to the
synthesis of enzymes and regulatory proteins necessary for DNA synthesis during
S phase and are mainly regulated by G1 cyclin-dependent kinase–cyclin
complexes (G1CdkC). Complexes of G1CdkC induce degradation of the S phase
inhibitors in late G1. Release of the S phase CdkC complex subsequently
stimulates entry into the S phase. Variations in the duration of the G1 phase of
the cell cycle, ranging from less than 8 hours to longer than 100 hours,
account for the different generation times exhibited by different types of
cells.
S Phase
The nuclear DNA content of the cell is duplicated during the S phase of the
cell cycle. The S-phase CdkC complex activates proteins of the DNA
prereplication complexes that assemble on DNA replication origins during G1.
The prereplication complex activates initiation of DNA replication and inhibits
the assembly of new prereplication complexes. This inhibition ascertains that each
chromosome is replicated only once during the S phase.
G2 Phase
RNA and protein synthesis occurs during the G2 phase of the cell cycle. The
burst of biosynthetic activity provides the substrates and enzymes to meet the
metabolic requirements of the two daughter cells. Another important event that
occurs during the G2 phase of the cell cycle is the repair of errors of DNA
replication that may have occurred during the S phase. Failure to detect and
correct these genetic errors can result in a broad spectrum of adverse
consequences for the organism and the individual cell (1). Defects in the DNA
repair mechanism are associated with an increased incidence of cancer. Mitotic
CdkC complexes are synthesized during the S and G2 phases, but are inactive
until DNA synthesis is completed.
M Phase
Nuclear–chromosomal division occurs during the mitosis or M phase. During
236this phase, the cellular DNA is equally distributed to each of the daughter cells.
Mitosis provides a diploid (2n) DNA complement to each somatic daughter cell.
Following mitosis, eukaryotic mammalian cells contain diploid DNA, reflecting a
karyotype that includes 44 somatic chromosomes and an XX or XY sex
chromosome complement. Exceptions to the diploid cellular content include
hepatocytes (4n) and the functional syncytium of the placenta.
Mitosis is divided into prophase, metaphase, anaphase, and telophase.
Mitotic CdkC complexes induce chromosome condensation during the prophase,
assembly of the mitotic spindle apparatus, and alignment of the chromosomes
during the metaphase. Activation of the anaphase promoting complex (APC)
leads to inactivation of the protein complexes that connect sister chromatids
during metaphase, permitting the onset of anaphase. During anaphase, sister
chromatids segregate to opposite spindle poles. The nuclear envelope breaks
down into multiple small vesicles early in mitosis and reforms around the
segregated chromosomes as they decondense during telophase. Cytokinesis is the
process of division of the cytoplasm that segregates the endoplasmic reticulum
and the Golgi apparatus during mitosis. After completion of mitosis, cells enter
the G1 phase and either reenter the cell cycle or remain in G0.
Ploidy
After meiosis, germ cells contain a haploid (1n) genetic complement. After
fertilization, a 46,XX or 46,XY diploid DNA complement is restored.
Restoration of the normal cellular DNA content is crucial to normal function.
Abnormalities of cellular DNA content cause distinct phenotypic
abnormalities, as exemplified by a hydatidiform molar pregnancy (see
Chapter 39). In a complete hydatidiform mole, an oocyte without any
nuclear genetic material (e.g., an empty ovum) is fertilized by one sperm. The
haploid genetic content of the fertilized ovum is duplicated, and the diploid
cellular DNA content is restored, resulting in a homozygous 46,XX gamete.
Less often, a complete hydatidiform mole results from the fertilization of an
empty ovum by two sperm, resulting in a heterozygous 46,XX or 46,XY gamete.
In complete molar pregnancies, the nuclear DNA is usually paternally derived,
embryonic structures do not develop, and trophoblast hyperplasia occurs. Rarely,
complete moles are biparental. This karyotype seems to be found in patients with
recurrent hydatidiform moles and is associated with a higher risk of persistent
trophoblastic disease.
A partial hydatidiform mole follows the fertilization of a haploid ovum by
two sperm, resulting in a 69 XXX, 69 XXY, or 69 XYY karyotype. A partial
mole contains paternal and maternal DNA, and both embryonic and placental
237development occur. Both the 69 YYY karyotype and the 46 YY karyotype are
incompatible with embryonic and placental development. These observations
demonstrate the importance of maternal genetic material, in particular the X
chromosome, in normal embryonic and placental development.
In addition to total cellular DNA content, the chromosome number is an
important determinant of cellular function. Abnormalities of chromosome
number, which often are caused by nondisjunction during meiosis, result in wellcharacterized clinical syndromes such as trisomy 21 (Down syndrome), trisomy
18, and trisomy 13.
Genetic Control of the Cell Cycle
Cellular proliferation must occur to balance normal cell loss and maintain
tissue and organ integrity. This process requires the coordinated expression of
many genes at discrete times during the cell cycle. In the absence of growth
factors, cultured mammalian cells are arrested in the G0 phase. With the addition
of growth factors, these quiescent cells pass through the so-called restriction point
14 to 16 hours later and enter the S phase 6 to 8 hours thereafter. The restriction
point or the G1/S boundary marks the point at which a cell commits to
proliferation. A second checkpoint is the G2/M boundary, which marks the
point at which repair of any DNA damage must be completed (2). To
successfully complete the cell cycle, a number of cell division cycle (cdc) genes
are activated.
Cell Division Cycle Genes
Among the factors that regulate the cell cycle checkpoints, proteins encoded
by the cdc2 family of genes and the cyclin proteins appear to play
particularly important roles (3). Growth factor–stimulated mammalian cells
express early- or delayed-response genes, depending on the chronologic sequence
of the appearance of specific RNAs. The early- and delayed-response genes act as
nuclear transcription factors and stimulate the expression of a cascade of other
genes. Early-response genes such as c-Jun and c-Fos enhance the transcription of
delayed-response genes such as E2Fs. E2F transcription factors are required for
the expression of various cell cycle genes and are functionally regulated by the
retinoblastoma (Rb) protein. Binding of Rb to E2F converts E2F from a
transcriptional activator to a repressor of transcription. Phosphorylation of Rb
inhibits its repressing function and permits E2F-mediated activation of genes
required for entry into the S phase. Cdk4-cyclin D, Cdk6-cyclin D, and Cdk2-
cyclin E complexes cause phosphorylation of Rb, which remains phosphorylated
throughout the S, G2, and M phases of the cell cycle. After completion of mitosis,
238a decline of the level of Cdk-cyclins leads to dephosphorylation of Rb by
phosphatases and, consequently, an inhibition of E2F in the early G1 phase.
Cdks are under evaluation as targets for cancer treatments because they are
frequently overactive in cancer disease and Cdk-inhibiting proteins are
dysfunctional. The Cdk4 inhibitor P1446A-05, for example, specifically inhibits
Cdk4-mediated G1-S phase transition, arresting cell cycling and inhibiting cancer
cell growth (4). SNS-032 selectively binds to Cdk2, -7, and -9, preventing their
phosphorylation and activation and subsequently preventing cell proliferation.
As cells approach the G1-S phase transition, synthesis of cyclin A is initiated.
The Cdk2-cyclin A complex can trigger initiation of DNA synthesis by
supporting the prereplication complex. The cyclin-dependent kinase 1 (Cdk1)
protein, formerly called p34 cdc2, and specific cyclins (namely cyclin B1) form a
complex heterodimer referred to as mitosis-promoting factor (5), which catalyzes
protein phosphorylation and drives the cell into mitosis. Cdk1 assembles with
cyclin A and cyclin B in the G2 phase and promotes the activity of the mitosis
promoting factor (MPF). Mitosis is initiated by activation of the cdc gene at the
G2-M checkpoint (6). After the G2-M checkpoint is passed, the cell undergoes
mitosis. In the presence of abnormally replicated chromosomes, progression past
the G2-M checkpoint does not occur.
The p53 tumor suppressor gene participates in cell cycle control. Cells
exposed to radiation therapy exhibit an S-phase arrest that is accompanied by
increased expression of p53. This delay permits the repair of radiation-induced
DNA damage. In the presence of p53 mutations, the S-phase arrest that normally
follows radiation therapy does not occur (7). The wild type p53 gene can be
inactivated by the human papillomavirus (HPV) E6 protein, preventing S-phase
arrest in response to DNA damage.
Apoptosis
[1] The regulation and maintenance of normal tissue requires a balance
between cell proliferation and programmed cell death, or apoptosis. When
proliferation exceeds programmed cell death, the result is hyperplasia. When
programmed cell death exceeds proliferation, the result is atrophy. Programmed
cell death is a crucial concomitant of normal embryologic development. This
mechanism accounts for deletion of the interdigital webs, palatal fusion, and
development of the intestinal mucosa (8). Programmed cell death is an important
phenomenon in normal physiology. The reduction in the number of endometrial
cells following alterations in steroid hormone levels during the menstrual cycle is,
in part, a consequence of programmed cell death (9). In response to androgens,
granulosa cells undergo programmed cell death (e.g., follicular atresia) (10).
239Programmed cell death, or apoptosis, is an energy-dependent, active
process that is initiated by the expression of specific genes. This process is
distinct from cell necrosis, although both mechanisms result in a reduction in the
total cell number. In programmed cell death, cells shrink and undergo
phagocytosis. Conversely, groups of cells expand and lyse when undergoing cell
necrosis. This process is energy independent and results from noxious stimuli.
Programmed cell death is triggered by a variety of factors, including intracellular
signals and exogenous stimuli such as radiation exposure, chemotherapy, and
hormones. Cells undergoing programmed cell death may be identified on the
basis of histologic, biochemical, and molecular biologic changes. Histologically,
apoptotic cells exhibit cellular condensation and fragmentation of the nucleus.
Biochemical correlates of impending programmed cell death include an increase
in transglutaminase expression and fluxes in intracellular calcium concentration
(11).
Programmed cell death emerged as an important factor in the growth of
neoplasms. Historically, neoplastic growth was characterized by uncontrolled
cellular proliferation that resulted in a progressive increase in tumor burden. It is
recognized that the increase in tumor burden associated with progressive
disease reflects an imbalance between cell proliferation and cell death. Cancer
cells fail to respond to the normal signals to stop proliferating, and they may fail
to recognize the physiologic signals that trigger programmed cell death.
MODULATION OF CELL GROWTH AND FUNCTION
The normal cell exhibits an orchestrated response to the changing extracellular
environment. The three groups of substances that signal these extracellular
changes are steroid hormones, growth factors, and cytokines. The capability to
respond to these stimuli requires a cell surface recognition system, intracellular
signal transduction, and nuclear responses for the expression of specific genes in
a coordinated fashion. [2] Among the genes that participate in the control of
cell growth and function, proto-oncogenes and tumor suppressor genes are
particularly important. More than 100 proto-oncogene products that contribute
to growth regulation have been identified (Table 6-1) (12). As a group, protooncogenes exert positive effects upon cellular proliferation. In contrast, tumor
suppressor genes exert inhibitory regulatory effects on cellular proliferation
(Table 6-2).
Steroid Hormones
Steroid hormones play a crucial role in reproductive biology and in general
physiology. Among the various functions, steroid hormones influence pregnancy,
240cardiovascular function, bone metabolism, and an individual’s sense of wellbeing. The action of steroid hormones is mediated via extracellular signals to the
nucleus to affect a physiologic response.
Estrogens exert a variety of effects on growth and development of different
tissues. The effects of estrogens are mediated via estrogen receptors (ER),
intracellular proteins that function as ligand-activated transcription factors
and belong to the nuclear receptor superfamily (13). Two mammalian ERs
have been identified: ERα and ERβ. The structure of both receptors is similar
and consists of 6 domains named A through F from the N- to C-terminus,
encoded by 8 to 9 exons (14). Domains A and B are located at the N-terminus and
contain an agonist-independent transcriptional activation domain (activation
function 1 [AF-1]). The C domain is a highly conserved central DNA-binding
domain composed of two zinc fingers through which ER interacts with the major
groove and the phosphate backbone of the DNA helix. The C-terminus of the
protein contains domains E and F and functions as ligand-binding domain (LBD–
domain E) and AF-2–domain F (Fig. 6-3).
Table 6-1 Proto-Oncogenes
Proto-Oncogenes Gene Product/Function
Growth Factors
Fibroblast growth factor
fgf-5
Sis Platelet-derived growth factor β
hst, int-2
Transmembrane Receptors
erbB Epidermal growth factor (EGF)
receptor
HER2/neu EGF-related receptor
Fms Colony-stimulating factor (CSF)
receptor
Kit Stem cell receptor
Trk Nerve growth factor receptor
241Inner-Membrane Receptor
bcl-2
H-ras, N-ras, K-ras
fgr, lck, src, yes
Cytoplasmic Messengers
Crk
cot, plm-1, mos, raf/mil
Nuclear DNA-Binding Proteins
erb-B1
jun, ets-1, ets-2, fos, gil-1, rel, ski, vav
lyl-1, maf, myb, myc, L-myc, N-myc,
evi-1
Table 6-2 Tumor Suppressor Genes
p53 Mutated in as many as 50% of solid tumors
Rb Deletions and mutations predispose to retinoblastoma
PTEN Dual specificity phosphatase that represses PI3-kinase/Akt pathway
activation with negative effect on cell growth
P16INK4a Binds to cyclin-CDK4 complex inhibiting cell cycle progression
FHIT Fragile histidine triad gene with tumor suppressor function via
unknown mechanisms
WT1 Mutations are correlated with the Wilms tumor
NF1 Neurofibromatosis gene
APC Associated with colon cancer development in patients with familial
adenomatous
Activation of transcription via the ER is a multistep process. The initial
step requires activation of the ER via various mechanisms (Fig. 6-4). For
242example, estrogens such as 17β-estradiol can diffuse into the cell and bind to the
LBD of the ER. Upon ligand binding, the ER undergoes conformational changes
followed by a dissociation of various bound proteins, mainly heat shock proteins
90 and 70 (Hsp90 and Hsp70). Activation of the ER also requires phosphorylation
by several protein kinases, including casein kinase II, PKA, and components of
the Ras/MAPK (mitogen-activated protein kinase) pathway (14). Four
phosphorylation sites of the ER are clustered in the NH2 terminus with the AF-1
region.
FIGURE 6-3 Structure of the two mammalian estrogen receptors. ERα (595 amino acids)
and ERβ (530 amino acids) consist of six domains (A to F from the N- to C-terminus).
Domains A and B at the N-terminus contain an agonist-independent transcriptional
activation domain (activation function 1, or AF-1). The C domain is the central DNAbinding sequence (DBD). Domains E and F function as the ligand-binding domain (LBD)
and activation function 2 (AF-2). Also shown is the structure of the ER ligand 17β-
estradiol.
243FIGURE 6-4 Activation of estrogen receptor–mediated transcription. Intracellular
estrogen receptor signaling is mediated via different pathways. A: 17β-Estradiol diffuses
through the cell membrane and binds to cytoplasmic ER. The ER is subsequently
phosphorylated, undergoes dimerization, and binds to the estrogen response element (ERE)
on the promoter of an estrogen responsive gene. B: Estrogen ligand binds to membranebound ER and activates the mitogen-activated protein kinase (MAPK) pathways that
support ER-mediated transcription. C: Binding of cytokines such as insulinlike growth
factor (IGF) or epidermal growth factor to their membrane receptor can cause activation of
protein kinases like PKA, which subsequently activates ER by phosphorylation.
The activated ER elicits a number of different genomic as well as
244nongenomic effects on intracellular signaling pathways. The classical steroid
signaling pathway involves binding of the activated ER to an estrogen responsive
element (ERE) on the genome as homodimers and subsequent stimulation of
transcription (15). The minimal consensus sequence for the ERE is a 13 bp
palindromic inverted repeat (IR) and is defined as 5′-GGTCAnnnTGACC-3′.
Genes that are regulated by activated ERs include early gene responses such as cmyc, c-fos, and d-jun, and genes encoding for growth factors such as insulin
growth factor (IGF-1 and IGF-2), epidermal growth factor (EGF), transforming
growth factor α (TGF-α), and colony-stimulating factor (CSF-1).
Various ligands with different affinities to the ER were developed and are
called selective estrogen receptor modulators (SERMs). Tamoxifen, for
example, is a mixed agonist/antagonist for ERα, but it is a pure antagonist
for ERβ. The ERβ receptor is ubiquitously expressed in hormone-responsive
tissues, whereas the expression of ERα fluctuates in response to the hormonal
milieu. The cellular and tissue effects of an estrogenic compound appear to reflect
a dynamic interplay between the actions of these ER isoforms. These observations
underscore the complexity of estrogen interactions with both normal and
neoplastic tissues. Mutations of hormone receptors and their functional
consequences illustrate their important contributions to normal physiology.
Growth Factors
Growth factors are polypeptides that are produced by a variety of cell types
and exhibit a wide range of overlapping biochemical actions. Growth factors
bind to high-affinity cell membrane receptors and trigger complex positive and
negative signaling pathways that regulate cell proliferation and differentiation
(16). In general, growth factors exert positive or negative effects upon the cell
cycle by influencing gene expression related to events that occur at the G1-S
cell cycle boundary.
Because of their short half-life in the extracellular space, growth factors
act over limited distances through autocrine or paracrine mechanisms. In the
autocrine loop, the growth factor acts on the cell that produced it. The paracrine
mechanism of growth control involves the effect of growth factors on another cell
in proximity. Growth factors that play an important role in female reproductive
physiology are listed in Table 6-3. The biologic response of a cell to a specific
growth factor depends on a variety of factors, including the cell type, the cellular
microenvironment, and the cell cycle status.
The regulation of ovarian function occurs through autocrine, paracrine,
and endocrine mechanisms. The growth and differentiation of ovarian cells are
particularly influenced by the insulinlike growth factors (IGF) (Fig. 6-5). IGFs
245amplify the actions of gonadotropin hormones on autocrine and paracrine growth
factors found in the ovary. IGF-1 acts on granulosa cells to cause an increase in
cAMP, progesterone, oxytocin, proteoglycans, and inhibin. On theca cells, IGF-1
causes an increase in androgen production. Theca cells produce tumor necrosis
factor α (TNF-α) and EGF, which are regulated by follicle-stimulating hormone
(FSH). EGF acts on granulosa cells to stimulate mitogenesis. Follicular fluid
contains IGF-1, IGF-2, TNF-α, TNF-β, and EGF. Disruption of these autocrine
and paracrine intraovarian pathways may be the basis of polycystic ovarian
disease, disorders of ovulation, and ovarian neoplastic disease.
TGF-β activates intracytoplasmic serine threonine kinases and inhibits cells in
the late G1 phase of the cell cycle (17). It appears to play an important role in
embryonic remodeling. Mullerian-inhibiting substance (MIS), which is
responsible for regression of the mullerian duct, is structurally and functionally
related to TGF-β (18). TGF-α is an EGF homologue that binds to the EGF
receptor and acts as an autocrine factor in normal cells. As with EGF, TGF-α
promotes the entry of G0 cells into the G1 phase of the cell cycle. The role of
growth factors in endometrial growth and function was the subject of several
reviews (17,19). Similar to the ovary, autocrine, paracrine, and endocrine
mechanisms of control also occur in the endometrial tissue.
Table 6-3 Growth Factors That Play Important Roles in Female Reproductive
Physiology
246Intracellular Signal Transduction
[3] Growth factors trigger intracellular biochemical signals by binding to cell
membrane receptors. In general, these membrane-bound receptors are
protein kinases that convert an extracellular signal into an intracellular
signal. The interaction between growth factor ligand and its receptor results in
receptor dimerization, autophosphorylation, and tyrosine kinase activation.
Activated receptors in turn phosphorylate substrates in the cytoplasm and trigger
the intracellular signal transduction system (Fig. 6-6). The intracellular signal
transduction system relies on serine threonine kinases, src-related kinases, and G
proteins. Intracellular signals activate nuclear factors that regulate gene
expression. Many of the proteins that participate in the intracellular signal
transduction system are encoded by proto-oncogenes that are divided into
subgroups based on their cellular location or enzymatic function (Fig. 6-7)
(20).
247FIGURE 6-5 The regulation of ovarian function occurs through autocrine, paracrine, and
endocrine mechanisms.
248249FIGURE 6-6 Pathways of intracellular signal transduction.
The raf and mos proto-oncogenes encode proteins with serine threonine kinase
activity. These kinases integrate signals originating at the cell membrane with
those that are forwarded to the nucleus (21). Protein kinase C (PKC) is an
important component of a second messenger system that exhibits serine threonine
kinase activity. This enzyme plays a central role in phosphorylation, which is a
general mechanism for activating and deactivating proteins. It also plays an
important role in cell metabolism and division (22).
The Src family of tyrosine kinases is related to PKC and includes protein
products encoded by the src, yes, fgr, hck, lyn, fyn, lck, alt, and fps/fes protooncogenes. These proteins bind to the inner cell membrane surface.
The G proteins are guanyl nucleotide-binding proteins. The heterotrimeric, or
large G proteins, link receptor activation with effector proteins such as adenyl
cyclase, which activates the cAMP-dependent, kinase-signaling cascade (23). The
monomeric or small G proteins, encoded by the Ras proto-oncogene family, are
designated p21 and are particularly important regulators of mitogenic signals. The
p21 Ras protein exhibits guanyl triphosphate (GTP) binding and GTPase activity.
Hydrolysis of GTP to guanyl diphosphate (GDP) terminates p21 Ras activity. The
p21 Ras protein influences the production of deoxyguanosine and inositol
phosphate (IP) 3, arachidonic acid production, and IP turnover.
The phosphoinositide 3 (PI3) kinase can be activated by various growth factors
like platelet-derived growth factor (PDGF) or IGF results. Activation of PI3
kinase results in an increase of intracellular, membrane-bound lipids,
phosphatidylinositol-(3),(4)-diphosphate (PIP2), and phosphatidylinositol-(3),(4),
(5)-triphosphate (PIP3). The Akt protein is subsequently phosphorylated by PIP3-
dependent kinases (PDK) for full activation. Activated Akt is released from the
membrane and elicits downstream effects that lead to an increase in cell
proliferation, prevention of apoptosis, invasiveness, drug resistance, and
neoangiogenesis (24). The PTEN (phosphatase and chicken tensin homologue
deleted on chromosome 10) protein is an important factor in the PI3 kinase
pathway, because it counteracts the activation of Akt by dephosphorylating PIP3.
Cells with mutated tumor suppressor gene PTEN and lack of functional PTEN
expression display an increased proliferation rate and decreased apoptosis,
possibly supporting the development of a malignant phenotype. PTEN frequently
is mutated in endometrioid adenocarcinoma. Furthermore, lack of functional
PTEN expression was described in endometriosis.
The mammalian target of rapamycin (mTOR) is regulated by the PI3 kinase
pathway. mTOR is a serine/threonine protein kinase that regulates a variety of
250cellular processes, including proliferation, motility, and translation (25). mTOR
integrates the input from various upstream pathways, including insulin and
growth factors like IGF proteins. The mTOR pathway provides important survival
signals for cancer cells and therefore was one focus of targeted drug development
(26). For example, rapamycin inhibits mTOR by associating with its intracellular
receptor FKBP12. Derivatives of rapamycin like everolimus (RAD001) and
temsirolimus (CCI779) showed promising results in clinical trials (27).
FIGURE 6-7 Proto-oncogenes are divided into subgroups based on their cellular location
or enzymatic function.
mTOR functions as the catalytic subunit of two different protein complexes.
Among the proteins associated with mTOR complex 1 (mTORC1) is mTOR, the
regulatory associated protein of mTOR (Raptor), and PRAS40. This complex
functions as a nutrient and energy sensor and controls protein synthesis (28).
mTORC1 is activated by insulin, growth factors, amino acids and oxidative stress,
low nutrient levels, reductive stress, and growth factor deprivation inhibit its
activity. In contrast, the mTOR complex 2 (mTORC2) contains, among others,
251mTOR, the rapamycin-insensitive protein Rictor, and mammalian stress-activated
protein kinase interacting protein 1 (mSIN1). mTORC2 regulates the cytoskeleton
and phosphorylates Akt (29). Its regulation is complex, but involves insulin,
growth factors, serum, and nutrient levels.
Expression of Genes and Proteins
Ovarian, endometrial, and cervical cancers have distinct molecular and genomic
profiles. Ovarian carcinomas, in particular the high-grade serous histologic
subtypes, are characterized by genomic instability with low frequencies of
conserved mutations (30). Despite substantial differences between the
gynecologic cancers, activation of the PI3K pathway is common.
Endometrial cancers demonstrate the highest frequency of PI3K pathway
alterations (31). The rate of alterations in PIK3CA range from 25% to 40% and in
the PIK3R1 from 15% to 25% (32). In addition to PI3K alterations, 30% to 90%
of endometrial cancers have impaired PTEN function, most often identified in
endometrioid histologies (33). PI3K inhibition may impair proliferation of
endometrial cancer cells. In addition, loss of PTEN or PIK3CA mutation may
serve as biomarkers associated with response to novel targeted agents that
antagonize PI3K (34). Studies utilizing patient-derived xenograft (PDX) models
of endometrial carcinomas, with and without alterations in PIK3CA, have
suggested that pan-PI3K inhibition may produce significant anti-tumor responses.
Most of the published data regarding PI3K pathway inhibitors in endometrial
cancer have been based on mTOR inhibition. Rapalog inhibitors (everolimus,
ridaforolimus, and temsirolimus) have been demonstrated to inhibit mTORC1
complex kinase activity. However, in clinical application, the response rates to
monotherapy have been low, ranging from 4% to 25%; the higher response rates
correlating with patients extensively pretreated with cytotoxic chemotherapy (35).
Combination treatment of everolimus and letrozole showed a 32% response rate
(11 of 35 patients) (36). These response rates and duration are similar to treatment
with alternating courses of megestrol acetate and tamoxifen, with an overall RR
of 27%. Based on these comparable response findings, a randomized study is
underway comparing these two treatment strategies (GOG-3007) (36).
Genetic alterations of the PI3K pathway have been identified in up to 70% of
ovarian cancers (37). Gain of function mutations in PIK3CA have been observed
in 4% to 12% of ovarian cancers (38). Ovarian clear cell and endometrioid
adenocarcinomas demonstrate a higher frequency of mutations with 33% to 40%
activating mutations in PIK3CA in clear cell carcinomas, and 12% to 20% of
endometrioid carcinomas (39).
In cervical cancer, amplifications and activating mutations of the PIK3CA gene
have been demonstrated in 23% to 36% of cases, with the highest mutation rate
252found in PIK3CA gene (40). The HPV oncogenic proteins, namely E6, and E7
enhance PI3K pathway signaling via direct activation of PI3K, as well as
downstream AKT and rpS6K. These proteins induce ligand-dependent signaling
indirectly through epidermal growth factor receptor (EGFR). Similar to ovarian
cancer, data pertaining to the use of targeted PI3K pathway therapy in cervical
carcinoma is limited, with studies underway.
Regulation of genetic transcription and replication is crucial to the normal
function of the daughter cells, the tissues, and ultimately the organism.
Transmission of external signals to the nucleus by way of the intracellular
signal transduction cascade culminates in the transcription of specific genes
and translation of the mRNA into proteins that ultimately affect the
structure, function, and proliferation of the cell.
The human genome project resulted in the determination of the sequence
of DNA of the entire human genome (41). With the completion of this
project, it appears that the human haploid genome contains 23,000 protein
coding genes. Sequencing the human genome is a major scientific achievement
that has opened the door for more detailed studies of structural and functional
genomics. Structural genomics involves the study of three-dimensional
structures of proteins based on their amino acid sequences. Functional
genomics provides a way to correlate the structure and function. Proteomics
involves the identification and cataloging of all proteins used by a cell, and
cytomics involves the study of cellular dynamics, including intracellular
system regulation and response to external stimuli. The transcriptome is the
set of all RNA molecules, including mRNA, rRNA, tRNA, and other noncoding
RNA produced in one or a population of cells. The transcriptome varies with
external environmental conditions and reflects the actively expressed genes. The
metabolome describes a set of small-molecule metabolites, including
hormones and signaling molecules, that are found in a single organism.
Similar to the transcriptome and proteome, the metabolome is subject to
rapid changes (42). The kinome of an organism describes a set of protein
kinases, enzymes that are crucial for phosphorylation reactions.
Cancer Genetics
Cancer is a genetic disease that results from a series of mutations in various
cancer genes. Uncontrolled cell growth occurs because of accumulation of
somatic mutations or the inheritance of one or more mutations through the
germline, followed by additional somatic mutations. The mutation in genes
that are directly involved in normal cellular growth and proliferation can
lead to the development of uncontrolled growth, invasion, and metastasis.
According to the Knudson hypothesis, which was first described in
253children with hereditary retinoblastoma, two hits or mutations within the
genome of a cell are required for a malignant phenotype to develop (43). In
hereditary cancers, the first hit is present in the genome of every cell. Only
one additional hit is necessary, therefore, to disrupt the correct function of
the second cancer gene allele. In contrast, sporadic cancers develop in cells
without hereditary mutations in the cancer predisposing alleles. In this case,
both hits must occur in a single somatic cell to disrupt both cancer gene
alleles (Fig. 6-8).
Most adult solid tumors require 5 to 10 rate-limiting mutations to acquire
the malignant phenotype. Among these mutations, some are responsible for
causing the cancer phenotype, whereas others might be considered bystander
mutations, as with, for example, the amplification of genes that are adjacent to an
oncogene. The most compelling evidence for the mutagenic tumor development
process is that the age-specific incidence rates for most human epithelial tumors
increase at roughly the fourth to eighth power of elapsed time.
Gatekeepers and Caretakers
[4] Cancer susceptibility genes are divided into “gatekeepers” and
“caretakers” (44). Gatekeeper genes control cellular proliferation and are
divided into oncogenes and tumor suppressor genes. In general, oncogenes
stimulate cell growth and proliferation, and tumor suppressor genes reduce
the rate of cell proliferation or induce apoptosis. Gatekeepers prevent the
development of tumors by inhibiting growth or promoting cell death.
Examples of gatekeeper genes include the tumor suppressor gene p53 and the
retinoblastoma gene.
Caretaker genes preserve the integrity of the genome and are involved in DNA
repair (stability genes). The inactivation of caretakers increases the likelihood of
persistent mutations in gatekeeper genes and other cancer-related genes. The
DNA mismatch repair (MMR) genes, MLH1, MSH2, and MSH6, are examples of
caretaker genes.
254FIGURE 6-8 Hereditary and sporadic cancer development based on the Knudson “twohit” genetic model. All cells harbor one mutant tumor suppressor gene allele in hereditary
cancer. The loss of the second allele results in the malignant phenotype. Sporadic cancers
develop in cells with normal genome, therefore requiring both alleles to be inactivated (two
hits).
Hereditary Cancer
Most cancers are caused by spontaneous somatic mutations. However, a
small percentage of cancers arise on a heritable genomic background. About
12% of all ovarian cancers and about 5% of endometrial cancers are
considered to be hereditary (45,46). Germline mutations require additional
mutations at one or more loci for tumorigenesis to occur. These mutations occur
via different mechanisms, for example, via environmental factors such as ionizing
radiation or mutations of stability genes. Characteristics of hereditary cancers
include diagnosis at a relatively early age and a family history of cancer,
usually of a specific cancer syndrome, in two or more relatives. Hereditary
cancer syndromes associated with gynecologic tumors are summarized in Table
6-4.
[4] Various cancer-causing genetic and epigenetic mechanisms are described.
On the genomic level, gain of function gene mutations can lead to a
conversion of proto-oncogenes into oncogenes, and loss of function gene
mutations can inactivate tumor suppressor genes. Epigenetic changes include
DNA methylation, which can cause inactivation of tumor suppressor gene
expression by preventing the correct function of the associated promoter
sequence. Collectively, these genetic and epigenetic changes are responsible for
the development of cancer characterized by the ability of cells to invade and
metastasize, grow independently of growth factor support, and escape from
255antitumor immune responses.
Oncogenes
Oncogenes comprise a family of genes that result from the gain of function
mutations of their normal counterparts, proto-oncogenes. The normal
function of proto-oncogenes is to stimulate proliferation in a controlled
context. Activation of oncogenes can lead to stimulation of cell proliferation
and development of a malignant phenotype. Oncogenes were initially
discovered through retroviral tumorigenesis. Viral infection of mammalian cells
can result in integration of the viral sequences into the proto-oncogene sequence
of the host cell. The integrated viral promoter activates transcription from the
surrounding DNA sequences, including the proto-oncogene. Enhanced
transcription of the proto-oncogene sequences results in the overexpression of
growth factors, growth factor receptors, and signal transduction proteins, which
result in stimulation of cell proliferation. One of the most important group of viral
oncogenes is the family of ras genes, which include c-H(Harvey)-ras, cK(Kirsten)-ras, and N(Neuroblastoma)-ras.
Tumor Suppressor Genes
[5] Tumor suppressor genes are involved in the development of most cancers
and are usually inactivated in a two-step process in which both copies of the
tumor suppressor gene are mutated or inactivated by epigenetic mechanisms
like methylation (47). The most commonly mutated tumor suppressor gene in
human cancers is p53 (48). The p53 protein regulates transcription of other
genes involved in cell cycle arrest such as p21. Upregulation of p53 expression is
induced by DNA damage and contributes to cell cycle arrest, allowing DNA
repair to occur. p53 also plays an important role in the initiation of apoptosis. The
most common mechanism of inactivation of p53 differs from the classic two-hit
model. In most cases, missense mutations that change a single amino acid in the
DNA-binding domain of p53 result in overexpression of nonfunctional p53
protein in the nucleus of the cell.
The identification of tumor suppressor genes was facilitated by positional
cloning strategies. The main approaches are cytogenetic studies to identify
chromosomal alterations in tumor specimens, DNA linkage techniques to localize
genes involved in inherited predisposition to cancer, and examination for loss of
heterozygosity (LOH) or allelic alterations among studies in sporadic tumors.
Comparative genomic hybridization (CGH) allows fluorescence identification of
chromosome gain and loss in human cancers within a similar experiment.
Table 6-4 Hereditary Cancer Syndromes Associated With Gynecologic Tumors
256Hereditary
Syndrome
Gene
Mutation
Tumor Phenotype
Li–Fraumeni
syndrome
TP53,
CHEK2
Breast cancer, soft tissue sarcoma, adrenal
cortical carcinoma, brain tumors
Cowden
syndrome,
Bannayan–
Zonana
syndrome
PTEN Breast cancer, hamartoma, glioma,
endometrial cancer
Hereditary
breast and
ovarian cancer
BRCA1,
BRCA2
Cancer of breast, ovary, fallopian tube
Hereditary
nonpolyposis
colorectal
cancer
(HNPCC)
MLH1,
MSH2,
MSH3,
MSH6,
PMS2
Cancer of colon, endometrium, ovary,
stomach, small bowel, urinary tract
Multiple
endocrine
neoplasia type
I
Menin Cancer of thyroid, pancreas, and pituitary,
ovarian carcinoid
Multiple
endocrine
neoplasia type
II
RET Cancer of thyroid and parathyroid,
pheochromocytoma, ovarian carcinoid
Peutz–Jeghers
syndrome
STK11 Gastrointestinal hamartomatous polyps,
tumors of the stomach, duodenum, colon,
ovarian sex cord tumor with annular tubules
(SCTAT)
Stability Genes
The third class of cancer genes is “stability genes,” which promotes
tumorigenesis in a different way from tumor suppressor genes or amplified
oncogenes. The main function of stability genes is the preservation of the
correct DNA sequence during DNA replication (caretaker function) (49).
Mistakes that are made during normal DNA replication or induced by exposure to
mutagens can be repaired by a variety of mechanisms that involve MMR genes,
257nuclear-type excision repair genes, and base excision repair genes. The
inactivation of stability genes potentially leads to a higher mutation rate in all
genes. However, only mutations in oncogenes and tumor suppressor genes
influence cell proliferation and confer a selective growth advantage to the mutant
cell. Similar to tumor suppressor genes, both alleles of stability genes must be
activated to cause loss of function.
Genetic Aberrations
Gene replication, transcription, and translation are imperfect processes, and
the fidelity is less than 100%. Genetic errors may result in abnormal structure
and function of genes and proteins. Genomic alterations such as gene
amplification, point mutations, and deletions or rearrangements were identified in
premalignant, malignant, and benign neoplasms of the female genital tract (Fig.
6-9).
258FIGURE 6-9 Genes can be amplified or undergo mutation, deletion, or rearrangement.
Amplification
Amplification refers to an increase in the copy number of a gene.
Amplification results in enhanced gene expression by increasing the amount of
template DNA that is available for transcription. Proto-oncogene amplification is
a relatively common event in malignancies of the female genital tract. The
HER2/neu proto-oncogene, also known as c-erbB-2 and HER2, encodes a 185
kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity. It belongs
to a family of transmembrane receptor genes that includes the EGF receptors
erbB-1, erbB-3, and erbB-4. HER2/neu interacts with a variety of different
259cellular proteins that increase cell proliferation. Overexpression of HER2/neu was
demonstrated in about 30% of breast cancers, 20% of advanced ovarian cancers,
and as many as 50% of endometrial cancers (50). The GOG-177 study (a
randomized prospective phase III study including patients with measurable stages
III to IV, or recurrent endometrial carcinoma and randomly assigned treatment
with either doxorubicin and cisplatin or doxorubicin, cisplatin, and paclitaxel
with G-CSF) also included tumor collection within the study parameters. A
review of the tumor specimens showed a 44% rate of HER2 overexpression (2+
or 3+ by immunohistochemical [IHC] staining) and 12% HER2 amplification by
fluorescence in situ hybridization (FISH) (51). Morrison et al. investigated HER2
expression and amplification in women with endometrial cancer, including a wide
variety of histologies. The investigation showed a correlation between HER2
expression and amplification and tumors of higher grade/stage, lymph node
positivity, and survival outcomes. Overall survival was significantly shorter in
patients with HER2 expression (median 5.2 years) and/or showed amplification
(median 3.5 years) versus those that did not (median 13 years) (52). The clinical
significance of HER2 overexpression or gene amplification continues to be
debated.
Point Mutations
Point mutations of a gene may remain without any consequence for the
expression and function of the protein (gene polymorphism). However, point
mutations can alter a codon sequence and subsequently disrupt the normal
function of a gene product. The ras gene family is an example of oncogeneencoded proteins that disrupt the intracellular signal transduction system
following point mutations. Transforming Ras proteins contain point mutations in
critical codons (i.e., codons [11, 12], [59], [61]) with decrease of GTPase activity
and subsequent expression of constitutively active Ras. Point mutations of the p53
gene are the most common genetic mutations described in solid tumors. These
mutations occur at preferential “hot spots” that coincide with the most highly
conserved regions of the gene. The p53 tumor suppressor gene encodes for a
phosphoprotein that is detectable in the nucleus of normal cells. When DNA
damage occurs, p53 can arrest cell cycle progression to allow the DNA to be
repaired or undergo apoptosis. The lack of function of normal p53 within a cancer
cell results in a loss of control of cell proliferation with inefficient DNA repair
and genetic instability. Somatic mutations of the p53 gene occur in approximately
50% of advanced ovarian cancers and 30% to 40% of endometrial cancers
(predominantly serous histology) but are uncommon in cervical cancer (53,54).
BRCA
260Point mutations in the BRCA1 and BRCA2 genes can alter the activity of
these genes and predispose to the development of breast and ovarian
cancer(s) (55). The frequency of BRCA1 and BRCA2 mutations in the general
population in the United States is estimated at 1:250. Specific founder mutations
were reported for various ethnic groups. For example, two BRCA1 mutations
(185delAG and 5382insC) and one BRCA2 mutation (6174delT) are found in
2.5% of Ashkenazi Jews of Central and Eastern European descent. Additional
founder mutations were described in other ethnic groups, including from the
Netherlands (BRCA1, 2804delAA and several large deletion mutations), Iceland
(BRCA2, 995del5), and Sweden (BRCA1, 3171ins5).
The BRCA proteins are involved in DNA repair. If DNA is damaged, for
example, by ionizing radiation or chemotherapy, the BRCA2 protein binds to the
RAD51 protein, which is central for the repair of double-stranded breaks via
homologous recombination. BRCA2 regulates the availability and activity of
RAD51 in this key reaction. Phosphorylation of the BRCA2/RAD51 complex
allows RAD51 to bind to the site of DNA damage and, in conjunction with
several other proteins, mediates repair of DNA by homologous recombination.
BRCA1 functions within a complex network of protein–protein interactions,
mediating DNA repair by homologous recombination and regulating transcription
via the BRCA1-associated surveillance complex (BASC).
Deletions and Rearrangements
Individuals carrying a germline BRCA1 or BRCA2 pathogenic mutation have an
elevated lifetime risk of developing breast and ovarian cancer(s). Based on a
meta-analysis by Chen et al., the 70-year risk of developing ovarian cancer is
40% and 18% for BRCA1 and BRCA2, respectively, and breast cancer is 57%
and 49% for BRCA1 and BRCA2, respectively (56). These germline mutations
are inherited in an autosomal-dominant fashion. Approximately 5% of breast and
20% of ovarian cancers arise in women carrying heterozygous germline mutations
in BRCA1 or BRCA2 (57). BRCA1 and BRCA2 are tumor suppressor genes.
Loss of nonmutated (wild-type) allele at either BRCA1 or BRCA2 locus, termed
locus-specific LOH, can be observed in tumors. Cells with loss of BRCA function
and resultant homologous recombination (HR)–based DNA repair deficiency
have enhanced sensitivity to DNA-damaging agents, namely cytotoxic platinumbased chemotherapies and targeted therapies (specifically inhibition of poly
[ADP-ribose] polymerases [PARPs]) (58). Although much research has
demonstrated that germline pathogenic mutations in BRCA1 or BRCA2 in
ovarian cancer can downregulate HR, and thereby increase a patient’s sensitivity
and response to platinum-based chemotherapy and PARP inhibition, there is less
evidence to support the potential impact of somatic sequence variants of BRCA
261function. Published studies have demonstrated that germline and somatic
pathogenic mutations in HR genes (BRCA1, BRCA2, ATM, BARD1, BRIP1,
CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D)
occur in 31% of patients with serous or nonserous ovarian cancer (59). Of these,
75% of germline HR mutations and 71% of somatic HR mutations were in BRCA
(60). The role of somatic pathogenic BRCA mutations in platinum-based
chemotherapy and PARP inhibitor sensitivity indicates that the number of
individuals with ovarian cancer who may benefit from such treatments are greater
than predicted by the frequency of germline pathogenic mutations alone.
Poly (ADP-Ribose) Polymerases
PARPs are vital components of DNA damage and repair pathways. The concept
of synthetic lethality is based on the idea that simultaneous loss of function of two
or more gene products can cause cell death, even if a deficiency in one is not
lethal (61). For example, a tumor cell without a lethal inactivating mutation of
BRCA DNA repair genes, might still be destroyed if also exposed to effects of
PARP inhibition. Given that HGSCs are associated with germline BRCA1 or
BRCA2 mutations, they remain the most suitable candidates for PARP inhibition.
DNA repair function is inherently decreased in tumor cells, compared with
normal cells. This discrepancy contributes to the high selectivity of PARP
inhibitors (61).
Three different PARP inhibitors have been approved by the FDA for the
treatment of ovarian cancer patients, each with a different clinical indication.
Olaparib and niraparib are indicated in patients with platinum-sensitive ovarian
cancer as maintenance therapy following response to platinum-based
chemotherapy for recurrent disease. Neither drug requires the presence of
germline or somatic mutations in BRCA1 or BRCA2 genes for this indication.
For the treatment of recurrent ovarian cancer with olaparib, patients must have
had three or more lines of chemotherapy and have a germline mutation in BRCA1
or BRCA2. Response rates in this patient population is about 30% based on
clinical trial data (62). Rucaparib is likewise approved for maintenance therapy in
patients with recurrent, platinum-sensitive ovarian cancer but requires the
presence of a germline or somatic BRCA1 or BRCA2 mutation (63). The most
common side effects with PARP inhibitors include hematologic toxicities
including thrombocytopenia and anemia, gastrointestinal side effects, in particular
nausea and fatigue.
Lynch Syndrome (Hereditary Nonpolyposis Colorectal Cancer Syndrome, HNPCC)
Lynch syndrome (LS) is an autosomal-dominant inherited disorder caused by
germline mutations in DNA MMR genes. Mutations are characterized by
262predisposition for cancers arising from the colon, rectum, small bowel,
endometrium, ovary, stomach, pancreas, renal pelvis, ureter, and brain (64). This
syndrome was first identified in patients with familial predisposition to
gastrointestinal cancers. Endometrial cancer often precedes colorectal and other
LS-associated malignancies. Women with LS have a 15% to 70% lifetime risk of
endometrial cancer and a 6% to 8% lifetime risk of ovarian cancer, with this wide
range accounting for a variety of histopathologic subtypes (65,66). Such a high
incidence has led to increased screening of endometrial carcinoma specimens for
abnormalities in the DNA MMR pathway. The most common and convincing
method of germline mutation analysis is focused on the four primary MMR genes
associated in LS, including MLH1, MSH2, MSH6, and PMS1. Mutations in
MSH2 and/or MLH1 account for 90% of heterozygous mutations, while MSH6
mutations account for most of the remaining cases (67). A smaller subset of
patients may carry mutations in the EPCAM gene, which can lead to Lynch
phenotype by way of hypermethylation and inactivation of MSH2 promoter. IHC
staining for loss of expression serves as the standard screening approach.
Microsatellite instability (MSI) testing is available as a substitute or additional
modality for abnormalities of the MMR system; however, this testing has been
shown to be less sensitive than IHC, primarily because of a failure to detect
several MSH6 germline mutation carriers (64).
Deletions and rearrangements reflect gross changes in the DNA template
that may result in the synthesis of a markedly altered protein product.
Somatic mutations may involve chromosomal translocations that result in
chimeric transcripts with juxtaposition of one gene to the regulatory region of
another gene. This mutation type is most commonly reported in leukemias,
lymphomas, and mesenchymal tumors. The Philadelphia chromosome in chronic
myeloid leukemia (CML), for example, is the result of a reciprocal translocation
between one chromosome 9 and one chromosome 22. The DNA sequence
removed from chromosome 9 contains the proto-oncogene c-ABL and inserts into
the BCR gene sequence on chromosome 22 (Philadelphia chromosome). The
resulting chimeric BCR-ABL gene product functions as a constitutively active
tyrosine kinase and stimulates cellular proliferation by such mechanisms as an
increase of growth factors.
Single-nucleotide polymorphism (SNP) describes a variation in the DNA
sequence (68). Single nucleotides in the genome differ between paired
chromosomes in either one individual or between two individuals. For example,
the sequences TGACTA and TCACTA contain one single change in the second
nucleotide from guanine (G) to cytosine (C). This results in a G and C allele for
this particular gene sequence. SNPs can occur within coding or noncoding
sequences of genes or in the intergenic regions. SNPs might not change the amino
263acid sequence of the protein that is produced (synonymous SNP) or produce a
different peptide (nonsynonymous SNPs). If SNPs are located in noncoding
regions, various other processes like gene splicing or transcription factor binding
might be affected.
The frequency of SNPs in a given population is provided by the minor allele
frequency. This frequency differs between ethnic groups and geographic
locations. SNPs were associated with various human diseases including cancer.
They influence the effect of drug treatment and responses to pathogens and
chemicals (69). SNPs are important for the comparison between genomes of
different populations, for example, providing information about the susceptibility
of a certain population to develop specific cancers (70).
THE CANCER GENOME ATLAS PROJECT
In 2006, the National Cancer Institute and the National Human Genome Research
Institute initiated TCGA project. The goal of the project is to provide a
comprehensive genomic characterization and sequence analysis of cancer
diseases. The initial phase included glioblastoma multiforme, lung, and ovarian
cancers (71,72).
TCGA is taking advantage of high-throughput genome analysis techniques,
including gene expression profiling, SNP genotyping, copy number variation
profiling, genomewide methylation profiling, microRNA profiling, and exon
sequencing (73). These data are accessible for researchers via TCGA webpage
(https://gdc.cancer.gov). The findings as pertaining to gynecologic malignancies
are summarized below.
Endometrial Carcinoma
An integrated genomic, transcriptomic, and proteomic characterization of 373
endometrial carcinomas (tumor samples and corresponding germline DNA) using
array- and sequencing-based technologies was performed (74). Uterine serous
tumors and 25% of high-grade endometrioid tumors exhibited extensive copy
number alterations, few DNA methylation changes, low ER and progesterone
receptor levels, and frequent TP53 mutations. The majority of endometrioid
tumors were identified as having frequent mutations in PTEN, CTNNB1,
PIK3CA, ARID1A, and KRAS, novel mutations in SWI/SNF chromatin
remodeling complex gene ARID5B, and few copy number alterations or TP53
mutations. In addition, 10% of endometrioid tumors were identified to have
markedly increased transversion mutation frequency and a newly identified
mutation in POLE. POLE is a catalytic subunit of DNA polymerase epsilon
involved in nuclear DNA replication and repair (75). These new findings classify
264endometrial carcinomas into four distinct categories: POLE ultramutated, MSI
hypermutated, copy-number low, and copy-number high. Somatic copy number
alterations (SCNAs) showed that most endometrioid tumors have few SCNAs,
most serous and serous-like tumors exhibit extensive SCNAs, and the extent of
SCNA roughly correlates with progression-free survival (74).
According to TCGA data, endometrial cancer has higher-frequency mutations
in the PI(3)K/AKT pathway than any other tumor type that has been studied.
Endometrioid endometrial carcinomas demonstrate parallels with characteristics
of colorectal carcinoma, specifically high frequency of MSI (40% and 11%,
respectively), POLE mutations (7% and 3%, respectively) which lends to
ultrahigh mutation rates, and frequent activation of WNT/CTNNB1 signaling.
Yet, endometrial carcinomas are unlike those of colorectal carcinomas as they
have unique KRAS and CTNNB1 mutations and a distinct means by which
pathway activation is achieved. Data pertaining to molecular characterization
showed 25% of tumors classified as high-grade endometrioid have a molecular
phenotype similar to uterine serous carcinomas, including frequent TP53
mutations and extensive SCNA.
Ovarian Carcinoma
TCGA review for ovarian cancer included 489 clinically annotated stages II to IV
high-grade serous ovarian cancer (HGSC) samples and corresponding normal
DNA. The summary of mutational spectrum includes high prevalence of
mutations in TP53 (present in at least 96% of samples), BRCA1, and BRCA2
(including both germline and somatic mutations) in 22% of the tumors (76).
HGSC demonstrates a high frequency of mutations in genes involved in
homologous recombination, and is hence considered a disease of high genomic
instability. The mutation spectrum in HGSC differs from those in other histologic
subtypes of ovarian cancer. Clear-cell ovarian cancer tumors have few TP53
mutations, yet frequent ARID1A and PIK3CA mutations (39). Endometrioid
ovarian cancers carry frequent CTNNB1, ARID1A, and PIK3CA mutations and a
lower rate of TP53; and mucinous ovarian cancer tumors have prevalent KRAS
mutations (77). Such differences between ovarian cancer subtypes demonstrate
the ongoing need for subtype-specific targeted therapeutic modalities.
Cervical Carcinoma
TCGA study collection included primary frozen cervical cancer tumor tissue and
blood samples from patients who had not previously received chemotherapy or
radiotherapy. Whole exome sequencing demonstrated the following significantly
mutated genes: ERBB3, CASP8, HLA-A, SHKBP1, and TGFBR2 (78).
265Amplifications and fusion events were reported involving the BCAR4 gene, a
metastasis-promoting IncRNA that enhances cell proliferation in estrogenresistant breast cancer by activating the HER2/3 pathway, which can be targeted
indirectly by lapatinib (a dual tyrosine kinase inhibitor that interrupts the HER2
and EGFR pathways) (78,79). Amplifications in CD274 and PDCD1LG2 were
identified, both of which have been associated with response to lapatinib.
Another identified novel genomic characteristic that further subclassifies cervical
cancers is a group of endometrial-like cervical cancers involved predominantly
with HPV-negative tumors and characterized by mutations in KRAS, ARID1A,
and PTEN. These proteins may serve as therapeutic targets.
IMMUNOLOGY
The immune system plays an essential part in host defense mechanisms, in
particular the response to infections and neoplastic transformation. Our increased
understanding of immune system regulation provides opportunities for the
development of novel immunotherapeutic and immunodiagnostic approaches.
Immunologic Mechanisms
The human immune system has the potential to respond to abnormal or
tumor cells in various ways. Some of these immune responses occur in an
innate or antigen-nonspecific manner, whereas others are adaptive or
antigen specific. Adaptive responses are specific to a given antigen. The
establishment of a memory response allows a more rapid and vigorous response
to the same antigen in future encounters. Various innate and adaptive immune
mechanisms are involved in responses to tumors, including cytotoxicity directed
to tumor cells mediated by cytotoxic T cells (CTLs), natural killer (NK) cells,
macrophages, and antibody-dependent cytotoxicity mediated by complementation
activation (80).
Adaptive or specific immune responses include humoral and cellular
responses. Humoral immune responses refer to the production of antibodies.
Antibodies are bifunctional molecules composed of a variable region with
specific antigen-binding sites, combined with a constant region that directs the
biologic activities of the antibody, such as binding to phagocytic cells or
activation of complement. Cellular immune responses are antigen-specific
immune responses mediated directly by activated immune cells rather than
by the production of antibodies. The distinction between humoral and cellular
responses is historical and originates from the experimental observation that
humoral immune function can be transferred by serum, whereas cellular immune
function requires the transfer of cells. Most immune responses include both
266humoral and cellular components. Several types of cells, including cells from
both the myeloid and lymphoid lineages, make up the immune system. Specific
humoral and cellular immune responses to foreign antigens involve the
coordinated action of populations of lymphocytes operating in concert with one
another and with phagocytic cells (macrophages). These cellular interactions
include direct cognate interactions involving cell-to-cell contact and cellular
interactions involving the secretion of and response to cytokines or lymphokines.
Lymphoid cells are found in lymphoid tissues, such as lymph nodes or spleen, or
in the peripheral circulation. The cells that make up the immune system originate
from stem cells in the bone marrow.
B Cells, Hormonal Immunity, and Monoclonal Antibodies
B lymphocytes synthesize and secrete antibodies. Mature, antigen-responsive B
cells develop from pre-B cells (committed B-cell progenitors) and differentiate to
become plasma cells, which produce large quantities of antibodies. Pre-B cells
originate from bone marrow stem cells in adults after rearrangement of
immunoglobulin genes from their germ cell configuration. Mature B cells express
cell surface immunoglobulin molecules that function as receptors for antigen.
Upon interaction with antigen, mature B cells respond to become
antibody-producing cells. The process requires the presence of appropriate cell–
cell stimulatory signals and cytokines. Monoclonal antibodies are directed against
a specific antigenic determinant. In contrast, polyclonal antibodies detect multiple
epitopes that might be presented by just one or a panel of proteins. The in vitro
production of monoclonal antibodies, pioneered by Kohler and Milstein in the
1970s, has become an invaluable diagnostic and therapeutic tool, particularly for
the management of malignancies (81). The tumor antigen CA125, for example,
was detected in a screen of antibodies generated against ovarian cancer cell lines.
A radioimmunoassay is widely used to measure CA125 in the serum of patients
with ovarian cancer and guide treatment decisions. Therapeutic approaches
utilized immunotoxin-conjugated monoclonal antibodies directed to human
ovarian adenocarcinoma antigens. These antibodies induce tumor cell killing and
can prolong survival in mice implanted with a human ovarian cancer cell line.
However, some obstacles limit the clinical use of monoclonal antibodies,
including tumor cell antigenic heterogeneity, modulation of tumor-associated
antigens, and cross-reactivity of normal host and tumor-associated antigens.
Unique tumor-specific antigens have not been identified. All tumor antigens have
to be considered as tumor-related antigens because they are expressed on
malignant and nonmalignant tissues. Because most monoclonal antibodies are
murine, the host’s immune system can recognize and respond to these foreign
mouse proteins. The use of the genetically engineered monoclonal antibodies
267composed of human-constant regions with specific antigen-reactive murine
variable regions can result in reduced antigenicity.
T Lymphocytes and Cellular Immunity
[6] T lymphocytes have a central role in the generation of immune responses
by acting as helper cells in both humoral and cellular immune responses and
by acting as effector cells in cellular responses. T-cell precursors originate in
the bone marrow and move to the thymus, where they mature into functional T
cells. During their thymic maturation, T cells that can recognize antigens in the
context of the major histocompatibility complex (MHC) molecules are selected,
while self-responding T cells are removed (82).
T cells can be distinguished from other types of lymphocytes by their cell
surface phenotype, based on the pattern of expression of various molecules,
and by differences in their biologic functions. All mature T cells express certain
cell surface molecules, such as the cluster determinant 3 (CD3) molecular
complex and the T-cell antigen receptor (TCR), found in close association with
the CD3 complex. T cells recognize antigens through the cell surface T-cell
antigen receptor. The structure and organization of this molecule are similar to
those of antibody molecules, which are the B-cell receptors for the antigen.
During T-cell development, the T-cell receptor gene undergoes gene
arrangements similar to those seen in B cells, but there are important differences
between the antigen receptors on B cells and T cells. The T-cell receptor is not
secreted, and its structure is somewhat different from that of antibody molecules.
The way in which the B-cell and T-cell receptors interact with antigens is quite
different. T cells can respond to antigens only when these antigens are presented
in association with MHC molecules on antigen-presenting cells. Effective antigen
presentation involves the processing of the antigen into small fragments of
peptide within the antigen-presenting cell and the subsequent presentation of
these fragments of antigen in association with MHC molecules expressed on the
surface of the antigen-presenting cell. T cells can respond to antigens only when
presented in this manner, unlike B cells, which can bind antigens directly, without
processing and presentation by antigen-presenting cells.
[7] There are two major subsets of mature T cells that are phenotypically
and functionally distinct: T-helper/inducer cells, which express the CD4 cell
surface marker, and the CTLs, which express the CD8 marker. The
expression of these markers is acquired during the passage of T cells through the
thymus. CD4 T cells can provide help to B cells, resulting in the production of
antibodies by B cells, and interact with antigen presented by antigen-presenting
cells in association with MHC class II molecules. CD4 T cells can act as helper
cells for other T cells. CD8 T cells include cells that are cytotoxic (cells that can
268kill target cells bearing appropriate antigens), and they interact with an antigen
presented on target cells in association with MHC class I molecules. These T cells
can inhibit the biologic functions of B cells or other T cells (80). Although the
primary biologic role of CTLs seems to be lysis of virus-infected autologous cells,
cytotoxic immune T cells can mediate the lysis of tumor cells directly.
Presumably, CTLs recognize antigens associated with MHC class I molecules on
tumor cells through their antigen-specific T-cell receptor, setting off a series of
events that ultimately results in the lysis of the target cell.
Monocytes and Macrophages
Monocytes and macrophages, which are myeloid cells, have important roles in
innate and adaptive immune responses; macrophages play a key part in the
generation of immune responses. T cells do not respond to foreign antigens unless
those antigens are processed and presented by antigen-presenting cells.
Macrophages (and B cells and dendritic cells) express MHC class II
molecules and are effective antigen-presenting cells for CD4 T cells. Helperinducer (CD4) T cells that bear a T-cell receptor of appropriate antigen and selfspecificity are activated by this antigen-presenting cell to provide help (various
factors—lymphokines—that induce the activation of other lymphocytes). In
addition to their role as antigen-presenting cells, macrophages play an important
part in innate responses by ingesting and killing microorganisms. Activated
macrophages, besides their many other functional capabilities, can act as
cytotoxic, antitumor killer cells.
Natural Killer Cells
NK cells are effector cells in an innate type of immune response: the nonspecific
killing of tumor cells and virus-infected cells. Therefore, NK activity represents
an innate form of immunity that does not require an adaptive, memory
response for optimal biologic function, but the antitumor activity can be
increased by exposure to several agents, particularly cytokines such as
interleukin-2 (IL-2). Characteristically, NK cells have a large granular
lymphocyte morphology. NK cells display a pattern of cell surface markers that
differs from those characteristic of T or B cells. NK cells can express a receptor
for the crystallizable fragment (Fc) portion of antibodies, and other NK-associated
markers. NK cells appear to be functionally and phenotypically heterogeneous,
when compared with T or B cells. The cells that can carry out antibody-dependent
cellular cytotoxicity, or antibody-targeted cytotoxicity, are NK-like cells.
Antibody-dependent cellular cytotoxicity by NK-like cells resulted in the lysis of
tumor cells in vitro. The mechanisms of this tumor cell killing are not clearly
understood, although close cellular contact between the effector cell and the target
269cell seems to be required.
Cytokines, Lymphokines, and Immune Mediators
Many events in the generation of immune responses (and during the effector
phase of immune responses) require or are enhanced by cytokines, which are
soluble mediator molecules (Table 6-5) (83,84). Cytokines are pleiotropic in
that they have multiple biologic functions that depend on the type of target cell or
its maturational state. Cytokines are heterogeneous in the sense that most
cytokines share little structural or amino acid homology. Cytokines are called
monokines if they are derived from monocytes, lymphokines if they are
derived from lymphocytes, interleukins if they exert their actions on
leukocytes, or interferons (IFNs) if they have antiviral effects. They are
produced by a wide variety of cell types and seem to have important roles in
many biologic responses outside the immune response, such as inflammation or
hematopoiesis. They may be involved in the pathophysiology of a wide range of
diseases and show great potential as therapeutic agents in immunotherapy for
cancer. Although cytokines are a heterogeneous group of proteins, they share
some characteristics. For instance, most cytokines are low- to intermediatemolecular-weight (10 to 60 kDa) glycosylated-secreted proteins. They are
involved in immunity and inflammation, are produced transiently and locally
(they act in an autocrine and paracrine rather than an endocrine manner), are
extremely potent in small concentrations, and interact with high-affinity cellular
receptors that are specific for each cytokine. The cell surface binding of cytokines
by specific receptors results in signal transduction followed by changes in gene
expression and, ultimately, by changes in cellular proliferation or altered cell
behavior, or both. Their biologic actions overlap, and exposure of responsive cells
to multiple cytokines can result in synergistic or antagonistic biologic effects.
T-cell subsets characterized by the secretion of distinct patterns of cytokines
were identified. TH1 and TH2 are two helper T-cell subpopulations that
control the nature of an immune response by secreting a characteristic and
mutually antagonistic set of cytokines: clones of TH1 produce IL-2 and IFN-
γ, whereas TH2 clones produce IL-4, IL-5, and IL-10 (85). A similar
dichotomy between TH1- and TH2-type responses was reported in humans.
Human IL-10 inhibits the production of IFN-γ and other cytokines by human
peripheral blood mononuclear cells and by suppressing the release of cytokines
(IL-1, IL-6, IL-8, and TNF-α) by activated monocytes (84). IL-10 downregulates
class II MHC expression on monocytes, resulting in a strong reduction in the
antigen-presenting capacity of these cells. Together, these observations support
the concept that IL-10 has an important role as an immune-inhibitory cytokine.
270Additional T-cell subsets were identified, including Th17 cells and regulatory T
cells (Treg). Th17 cells are a distinct, pro-inflammatory T-cell subset, which is
functionally characterized by mediating protection against extracellular bacteria
and by its pathogenic role in autoimmune disorders (86,87). Th17 cells
characteristically produce IL-17, CXCL13 (a B-cell stimulatory chemokine), IL-
6, and TNF-α, in contrast to Th2 cells, which characteristically produce IL-4, IL-
5, IL-9, and IL-13, or Th1 cells, which produce IFN-γ (Fig. 6-2). Treg cells
constitute another subset of CD4-positive T cells that participates in the
maintenance of immunologic self-tolerance by actively suppressing the activation
and expansion of self-reactive lymphocytes. Treg cells are characterized by the
expression of CD25 (the IL-2 receptor chain) and the transcription factor FoxP3
(88,89). Treg cell activity is thought to be important in preventing the
development of autoimmune diseases. Removal of Treg may enhance immune
responses against infectious agents or cancer. Although much remains to be
learned about the role of Treg activity in antitumor immunity, it is clear that such
cells may play a role in modulating host responses to cancer. Much of the recent
research has been directed at the tumor microenvironment. Specifically, the tumor
microenvironment consists of several varieties of cell types that interact with
tumor cells to influence tumor initiation, growth, and metastasis (90). The tumor
microenvironment plays a key role in allowing tumor cells to evade immune
recognition and destruction.
Because epithelial cancers of the ovary usually remain confined to the
peritoneal cavity, even in the advanced stages of the disease, it was suggested
that the growth of ovarian cancer intraperitoneally could be related to a local
deficiency of antitumor immune effector mechanisms (91). Studies showed
that ascitic fluid from patients with ovarian cancer contained increased
concentrations of IL-10. Various other cytokines are seen in ascitic fluid obtained
from women with ovarian cancer, including IL-6, IL-10, TNF-α, granulocyte
colony-stimulating factor (G-CSF), and granulocyte-macrophage colonystimulating factor (GM-CSF). A similar pattern was seen in serum samples from
women with ovarian cancer with elevations of IL-6 and IL-10.
TNF-α is a cytokine that can be directly cytotoxic for tumor cells, increase
immune cell–mediated cellular cytotoxicity, activate macrophages, and
induce secretion of monokines. Other biologic activities of TNF-α include the
induction of cachexia, inflammation, and fever; it is an important mediator of
endotoxic shock.
Cytokines in Cancer Therapy
Cytokines are exceptionally pleiotropic with a wide array of biologic activities,
including some outside the immune system (92). Because some cytokines have
271direct or indirect antitumor and immune-enhancing effects, several of these
factors are used in the experimental treatment of cancer.
The precise roles of cytokines in antitumor responses have not been
elucidated. Cytokines can exert antitumor effects by many different direct or
indirect activities. It is possible that a single cytokine could increase tumor growth
directly by acting as a growth factor while at the same time increasing immune
responses directed toward the tumor. The potential of cytokines to increase
antitumor immune responses was tested in experimental adoptive immunotherapy
by exposing the patient’s peripheral blood cells or tumor-infiltrating lymphocytes
to cytokines such as IL-2 in vitro, thus generating activated cells with antitumor
effects that can be given back to the patient (93). Some cytokines can exert direct
antitumor effects. TNF can induce cell death in sensitive tumor cells.
The effects of cytokines on patients with cancer might be modulated by soluble
receptors or blocking factors. For instance, blocking factors for TNF and for
lymphotoxin were found in ascitic fluid from patients with ovarian cancer (93).
Such factors could inhibit the cytolytic effects of TNF or lymphotoxin and should
be taken into account in the design of clinical trials of intraperitoneal infusion of
these cytokines.
Table 6-5 Sources, Target Cells, and Biologic Activities of Cytokines Involved in
Immune Responses
272Cytokines have growth-increasing effects on tumor cells in addition to
inducing antitumor effects. They can act as autocrine or paracrine growth
factors for human tumor cells, including those of nonlymphoid origin. For
instance, IL-6 (which is produced by various types of human tumor cells) can act
as a growth factor for human myeloma, Kaposi sarcoma, renal carcinoma, and
epithelial ovarian cancer cells (94).
273Cytokines are of great potential value in the treatment of cancer, but because of
their multiple, even conflicting, biologic effects, a thorough understanding of
cytokine biology is essential for their successful use (93).
FACTORS THAT TRIGGER NEOPLASIA
Cell biology is characterized by considerable redundancy and functional overlap,
so a defect in one mechanism does not invariably jeopardize the function of the
cell. When a sufficient number of abnormalities in structure and function occur,
normal cell activity is jeopardized, and uncontrolled cell growth or cell death
results. Either end point may result from accumulated genetic mutations. Factors
are identified that enhance the likelihood of genetic mutations, jeopardize normal
cell biology, and may increase the risk of cancer.
Increased Age
Increasing age is considered the single most important risk factor for the
development of cancer (95). Cancer is diagnosed in as much as 50% of the
population by 75 years of age. It was suggested that the increasing risk of cancer
with age reflects the accumulation of critical genetic mutations over time, which
ultimately culminates in neoplastic transformation. The basic premise of the
multistep somatic mutation theory of carcinogenesis is that genetic or epigenetic
alterations of numerous independent genes result in cancer. Factors that are
associated with an increased likelihood of cancer include exposure to exogenous
mutagens, altered host immune function, and certain inherited genetic syndromes
and disorders.
Environmental Factors
A mutagen is a compound that results in a genetic mutation. A number of
environmental pollutants act as mutagens when tested in vitro. Environmental
mutagens usually produce specific types of mutations that can be differentiated
from spontaneous mutations. A carcinogen is a compound that can produce
cancer. It is important to recognize that all carcinogens are not mutagens and that
all mutagens are not necessarily carcinogens.
Smoking
Cigarette smoking is perhaps the most well-known example of mutagen
exposure that is associated with the development of lung cancer when the
exposure is of sufficient duration and quantity in a susceptible individual. An
association between cigarette smoking and cervical cancer has been recognized
274for decades. It was determined that the mutagens in cigarette smoke are
selectively concentrated in cervical mucus (96). It was hypothesized that
cigarette smoke acted as a mutagen when exposed to the proliferating epithelial
cells of the transformation zone, thus increasing the likelihood of DNA damage
and subsequent cellular transformation.
Others observed that HPV DNA is frequently inserted into the fragile
histidine triad (FHIT) gene in cervical cancer specimens. The FHIT is an
important tumor suppressor gene. Cigarette smoking might facilitate the
incorporation of HPV DNA into the FHIT gene with subsequent disruption
of correct tumor suppressor gene function.
Radiation
Radiation exposure can increase the risk of cancer. The biologic effects of
ionization surpass those of x-rays and gamma rays. Ionization of water molecules
creates hydroxyl radicals, which cause DNA strand breaks or base damage. Most
radiation-induced damage is repaired precipitously. However, misrepair may
cause point mutations, chromosome translocations, and gene fusions associated
with cancer initiation (97). Normally, radiation damage prompts an S-phase arrest
so that DNA damage is repaired. This requires normal p53 gene function. If DNA
repair fails, the damaged DNA is propagated to daughter cells following mitosis.
If a sufficient number of critical genes are mutated, cellular transformation may
result (98).
Immune Function
Systemic immune dysfunction was recognized for decades as a risk factor for
cancer. Patients infected with HIV who have a depressed CD4 cell count are
reported to be at increased risk of cervical dysplasia (99). Individuals who
underwent high-dose chemotherapy with stem cell support may be at increased
risk of developing a variety of solid neoplasms. These examples illustrate the
importance of immune function in host surveillance for transformed cells.
Another example of altered immune function that may be related to the
development of cervical dysplasia is the alteration in mucosal immune function
that occurs in women who smoke cigarettes (100). The Langerhans cell
population of the cervix is decreased in women who smoke. Langerhans cells are
responsible for antigen processing. It is postulated that a reduction in these cells
increases the likelihood of successful HPV infection of the cervix.
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