Cancer Biology & Therapy

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Molecular pathogenesis of cervical cancer

Okechukwu A. Ibeanu

To cite this article: Okechukwu A. Ibeanu (2011) Molecular pathogenesis of cervical cancer ,
Cancer Biology & Therapy, 11:3, 295-306, DOI: 10.4161/cbt.11.3.14686

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Published online: 01 Feb 2011.

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 REVIEW
Cancer Biology & Therapy 11:3, 295-306; February 1, 2011; 2011 Landes Bioscience
Molecular pathogenesis of cervical cancer
Okechukwu A. Ibeanu
Division of Gynecologic Oncology; Post-DoctoralFellow; Johns Hopkins University; Baltimore, MD USA
Key words: HPV, cervix, carcinogenesis, dysplasia, early, gene, vaccine
Introduction: Cervical cancer is a sexually transmitted disease
that results from infection with oncogenic types of human
papillomavirus (HPV). Oncogenic HPV DNA is found in over
95% of invasive cervical cancers worldwide. Cervical cancer
is a leading cause of cancer deaths in developing countries
because of high HPV infection rates and lack of comprehensive
cervical Pap smear testing of susceptible women. Vaccination
against HPV prevents the acquisition of cervical dysplastic
lesions among eligible women who have not already acquired
the vaccine-specific HPV types.
Methods: Literature review of the epidemiology, infection
cycle, viral gene function and current vaccines related to the
oncogenic types of the human papillomavirus (HPV).
Conclusion: Enhanced understanding of HPV and popula-
tion-based measures offer the best hope of limiting worldwide
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mortality due to cervical cancer. The development of thera-
peutic cervical cancer vaccines and/or virus-targeted drug
therapies would be a giant step forward.
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Introduction
Scientific studies over the past few decades have provided
definitive evidence that cervical cancer is a sexually transmit-
ted disease that results from infection with certain high-risk,
oncogenic types of the human papillomavirus. HPV DNA
is found in 99% of invasive cervical cancers worldwide.1,2
Furthermore, vaccination against HPV prevents the acquisition
of the precursor lesion of cervical cancer, high-grade squamous
intraepithelial lesions (HSILs), also termed high grade cervical
intraepithelial neoplasia (CIN2/3). While successful imple-
mentation of cervical cancer screening protocols and treat-
ment of HSIL have led to a decline in mortality in developed
areas of the world, cervical cancer continues to be a leading
cause of cancer related deaths in developing countries due to a
combination of the high prevalence of HPV infection and the
lack of widespread availability of cervical Papanicolau smear
(Pap smear) testing of susceptible women.3 Molecular stud-
ies of HPV carcinogenesis have revealed fundamental mecha-
nisms by which HPV replicates, transforms cells and evades
the immune system. Extensive epidemiologic data and clinical
trials have led to refinements in screening, including new clini-
cal tests for oncogenic HPV infection and treatment protocols
Correspondence to: Okechukwu A. Ibeanu; Email: oibeanu1@jhmi.edu
Submitted: 12/06/10; Accepted: 12/30/10
DOI: 10.4161/cbt.11.3.14686
www.landesbioscience.com Cancer Biology & Therapy 295
REVIEW
based on a better understanding of the natural course of HPV
infection and accompanying cervical premalignant disease.
This review aims to discuss the pathogenesis and immuno-
logic changes of HPV-related cervical cancer as well as the clini-
cal course of HPV infection in relation to current screening and
treatment guidelines. The role of the HPV early proteins will
be discussed as well as current clinical management of cervical
dysplasia.
Epidemiology of Human Papilloma Virus
Cervical cancer is the second most prevalent cancer seen in
women worldwide, with about 500,000 cases and over 270,000
deaths estimated annually.3,4 Sub-Saharan Africa, South America
and Southeast Asia have the highest prevalence of HPV with 25,
15 and 8% of women infected, respectively.3 In the United States,
cervical cancer is the 12th most common cancer in women with
11,000 cases and 3,500 deaths reported in 2008.5 The incidence
of cervical cancer in the United States has dropped by about 90%
over the past half century due to effective implementation of
cervical cancer screening and treatment protocols. Nonetheless,
considering that 80% of sexually active US adults will acquire
HPV during their lifetimes,4 HPV-related diseases will continue
to impose a considerable burden on healthcare resources for the
foreseeable future. Fortunately, the recent licensure of two pre-
ventive HPV vaccines provides an important opportunity to
eventually eliminate cervical cancer upon comprehensive vacci-
nation prior to sexual debut.
The central etiologic factor for the development of cervical
cancer is persistent infection with high-risk oncogenic HPV
types.6 Other recognized risk factors for cervical cancer are
related to the sexual acquisition of HPV as well as immune dys-
function, exposure to mutagens and hormonal factors. Studies
in twins suggest that genetic background typically plays a small
role in the development of cervical cancer. The most notable
risks are early age of sexual activity, multiple sexual partners,
exposure to other sexually transmitted diseases, cigarette smok-
ing, oral contraceptive use, human immunodeficiency virus
infection and immunosuppressive drug therapy.3 Poor access
to cancer screening services as well as non-compliance with
screening visits are social risk factors related to lower socio-
economic status and lower educational levels that are observed
more frequently among African American, American Indian
and Hispanic women compared to Caucasians in the United
States.3 Unfortunately, these same factors negatively impact
HPV vaccine implementation in these populations, given its
current high cost.
 Table 1. HPV classification into high- and low-risk types36
Low-risk types 6, 11, 40, 4244, 54, 61, 72, 81
High-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 82
HPV types and cervical disease. HPVs are small, non-
enveloped DNA tumor viruses. Their genomes of approximately
8 kb are covalent closed circular, encoding eight genes, from the
E1, E2, E4, E5, E6, E7, L1 and L2 open reading frames (ORFs).
The genome is wrapped around nucleosomes and coated by a
60 nm diameter T = 7 capsid. HPV is believed to have evolved
along with humans over many eons and productively infects only
humans, although many other animal species of papillomavirus
exist.7 Analyses of different HPV types have suggested a common
ancestry, which has diversified over time to include host-specific
types that have maintained a predilection for infecting basal
epithelial cells of the skin or mucosal epithelia, notably of the
genitalia and oropharynx.7,8 In the 1970s, hybridization studies
using DNA from skin and genital lesions suggested that there
were differences in the HPV types found in biopsy specimens of
skin and genital lesions. Subsequent experiments reported in the
1980s confirmed the existence of distinct HPV types in associa-
tion with dysplastic genital warts and invasive cervical cancers.6,9
There are at least one hundred different types of HPV classi-
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fied phylogenetically within the alpha, beta, gamma, delta and
mu genera based upon differences in their nucleotide sequences.
Alpha genus HPV types infect the genital and oropharnygeal
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mucosa exclusively and include the oncogenic HPV types associ-
ated with cervical cancer. HPV types are assigned on the basis
of a difference of at least 10% in the nucleotide sequence of the
L1 open reading frame. HPV variants differ generally by <3%
in their L1 sequences, and typically exhibit single-nucleotide
variation.8,10 Interestingly, HPV types are tissue tropic, and also
mostly lesion specific, meaning that pre-malignant and malignant
lesions of the cervix diagnosed in the clinical setting are associ-
ated with different HPV types than those seen in benign extra-
genital warts, for example. HPV types are classified as low-risk
and high-risk with regard to their clinical oncogenic potential.
The oncogenic potential is a function of the propensity of the
respective HPV associated lesions to progress to invasive cervical
cancer over time. It is important to remember that approximately
95% of invasive cervical cancers are squamous cell cancers, while
less than 5% are adenocarcinomas, and each is associated with a
different spectrum of HPV types. Several rare histologic subtypes
make up the remainder of cervical cancers.
The four major HPV types that cause genital disease are 6, 11,
16 and 18.9 HPV 6 was the initial genital type reported in 1982,
isolated from non-metastatic but locally invasive giant genital
warts (the rare Buschke-Lowenstein tumor), although it is typi-
cally found in benign genital warts (condyloma accuminata). In
1983, HPV 11 and HPV 16 were detected in cervical lesions.
HPV 11 was subsequently isolated consistently from laryngeal
papillomas and genital warts.6 Since then, multiple molecular
and epidemiologic studies have shown that HPV 6 and 11 are
generally seen in benign genital wart lesions (about 90%), and
are classed as low-risk types. In contrast, HPV types 16 and 18
296 Cancer Biology & Therapy Volume 11 Issue 3
are found in high-grade cervical dysplastic (also premalignant)
lesions and in the majority of frankly invasive cervical cancers.
Both HPV 16 and 18 are classed as high-risk oncogenic types
and are particularly likely to persist and progress from high-grade
premalignant cervical disease to invasive cancer. HPV types 31,
33, 45, 52 and 58 are also recognized as oncogenic, but are less
prevalent in cervical cancer.3,4,6-9 The less prevalent oncogenic
types will not be covered in this discussion. HPV 16 and HPV
18 are by far the most prevalent types in both symptomatic and
asymptomatic women, and are associated with about 5070%
and 2030% of invasive cervical cancers diagnosed worldwide,
respectively. Table 1 shows the classification by risk type for the
common genital HPV genotypes.
Four HPV 16 variants have been defined; European (E),
African (Af-1, Af-2), Asian American (Aa). Similarly, three HPV
18 variants are currently recognized; European (E), African (Af)
and Asian Amerindian (AsAi).4,7 The existence of these variants
is generally thought to reflect the migration and sexual behav-
ioral patterns of human populations over time. Together, HPV
16 and 18 cause over 95% of invasive squamous cell cervical can-
cers diagnosed. Cervical adenocarcinomas are mostly associated
with HPV 16, 18 and 45.4
Epidemiologic studies indicate that HPV genital-tropic types
are seen in both symptomatic and asymptomatic patients and
that the majority of infections spontaneously resolve. It is not
uncommon in the clinical setting to have multiple HPV types
present in the same patient. Multiple, simultaneous or sequential
genital infections can occur, and in adolescent girls it is often
difficult to distinguish between persistent infections and new
infections by different subtypes of HPV.11 While persistent infec-
tion with an oncogenic HPV is necessary for carcinogenesis, it
should be stressed that several other factors and poorly defined
molecular events drive the transformation of cervical epithelial
cells to a malignant state. Clinical studies have shown that such
transformation is associated with integration of the viral genome
and epithelial cell genetic instability that occurs over a long
period of HPV infection. The importance of genetic instability
is highlighted by the increased risk of cervical cancer in patients
with Fanconis anemia. Also significant are immunosuppression,
as seen in Human Immunodeficiency Virus (HIV) co-infection
and chronic steroid therapy following organ transplantation,
which increase the likelihood of persistence of an oncogenic HPV
infection and its sequelae.6,12 In fact, in HIV patients, cervical
cancer is a criterion for making a clinical diagnosis of Acquired
Immune Deficiency Syndrome (AIDS).
Immunologic Aspects of HPV Infection
The majority of immunocompetent individuals infected with
HPV are able to clear the viral infection and remain asymptom-
atic. It is, however, controversial whether the virus is truly elimi-
nated from the patient or suppressed to such an extent that it is
undetectable with current sampling and analytic approaches. In a
minority of patients, HPV infection persists and causes clinically
detectable lesions that can progress to invasive cancer over a long
time period, typically measured in years to decades.9,13 It is not
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Figure 1. Low-grade lesion, which extends into endocervical canal.
entirely clear why HPV infection persists in certain individuals,
but deficits in cellular immunity, such as treatment of solid organ
transplant patients with immunosuppressants and those co-
infected with HIV, are associated with increased persistence and
HPV related disease. Conversely, the prevention of new infec-
tions by vaccination with L1 viral like particles (VLPs) appears
to be primarily affected by neutralizing antibodies. In animal
models, passive transfer of serum immunoglobulins from L1
VLP-immunized animals is sufficient to confer protection upon
nave animals. Furthermore, the measurement of in vitro neu-
tralization titers in sera is the best available correlate to assess
protection in patients for vaccine types and also cross-protection
against non-vaccine types, although no minimal titer for protec-
tion has been defined as yet.
HPV Infection Cycle
As mentioned previously, HPV has a predilection for epithelial
cells of the cervix, which are stratified into a non-differentiated
basal monolayer and a suprabasal differentiated non-proliferating
epidermis. The basal layer sits above the basement membrane,
below which is the cervical stromal layer. Dividing immature
basal cells move upward through to the epidermal layer where
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they are shed as part of the natural process of epithelial matu-
ration. Traumatic micro-abrasions, such as occur during sexual
intercourse, expose the nave basal layer cells to HPV.14,15 Cell
entry is not well understood but is believed to be receptor-
mediated and multiple reports have implicated heparin sulfate
as a candidate molecule involved in this process.16,17 HPV rep-
lication is dependent upon and utilizes the normal replicative
machinery of the cervical cells, which is subverted by two viral
proteins, E1 and E2. The virus is maintained at typically ~100
episomal copies per basal cell and the initial infection triggers a
burst of viral replication up to this level.
Basal cells that are infected with HPV continue to divide and
each form two daughter cells containing viral genomic material.
One cell of the pair remains in the basal layer and retains its
dividing capacity, therefore acting as a repository for replication
of the virus, which requires active cell division to maintain its
life cycle. The other daughter cell continues upward through the
suprabasal layer, where it differentiates and eventually is shed
from the epithelial surface.16,18 In order to ensure that cervical
cells are maintained in a state of constant growth and division,
HPV early proteins are expressed, which stimulate and propagate
cell growth via the actions of the E5, E6 and E7 genes. Upon
cellular differentiation in the suprabasal layer, the viral genome is
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Figure 2. High-grade lesion with features of vascular punctation and mosaic pattern.
replicated to 10,000 or greater copies/cell, and expression of the
late viral genes E4, L1 and L2 is triggered. The L1 (major) and L2
(minor) proteins form the capsid structure around the genomic
material of the virus. Once this assembly is complete within the
cells, the mature viral particles are released from the epithelial
cells during terminal shedding from the epithelial surface.16,18,19
It is postulated that the E4 viral protein facilitates the release and
spread of HPV from the keratin cage within keratinocytes by col-
lapsing keratin filaments in the dying squames.
It should be noted that carcinogenic progression is not part
of the normal HPV life cycle, but rather a non-productive dead
end that is only associated with a small subset of virus types and
occurs only after a long period of infection. The vast majority of
infections are benign and self-limited.
HPV Virology
E1 and E2. The initial events after HPV achieves cell entry
and delivery of the genome to the nucleus trigger the expression
of the HPV early genes E1 and E2. The E1 and E2 genes acti-
vate viral replication through interaction with specific sequences
in the HPV genome origin of replication,18,19 and by co-opting
298 Cancer Biology & Therapy Volume 11 Issue 3
cellular DNA replication factors. By binding to sites that are
proximal to its promoter, E2 leads to the recruitment of E1 to
the HPV origin of replication. E1 binding to E2 increases stable
binding to the AT-rich sequences within the origin of replication
through the formation of a binding complex.
E2 contains about 360 amino acids and possesses a DNA
binding domain as well as a transactivation domain, and it
can be spliced into a truncated variant lacking the transactiva-
tion domain. Thus, through alternative splicing, E2 is capable
of both activation and repression of its target proteins in HPV.
Interestingly, E2 also downregulates E6 and E7. Since E2 expres-
sion is typically lost during viral integration and carcinogenic
progression, E6 and E7 are relieved of the suppressive influence
of E2, leading to elevated expression of these viral oncogenes.18,19
By repression of an early promoter, E2 self-regulates levels of
E1 and E2 transcription to maintain a stable viral copy number
(~102 /cell) in the basal epithelia. As keratinocyte differentiation
progresses in the upper layers of squamous epithelia, the tran-
scription of E1 and E2 switches to a different (late) promoter that
is not modulated by E2, thus allowing for vegetative replication
and increased (~104/cell) viral genomic copies. Following this,
expression of the capsid antigens leads to the formation of
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Figure 3. High-grade lesion with punctation and mosaicism also noted.
viral particles by encapsidation of the nucleosome-bound viral
genomes.
E5. As part of the early replication process, the E5 gene
is expressed. E5 is a small, hydrophobic, single membrane-
spanning protein that complexes with platelet derived growth
factor receptor and epidermal growth factor receptor to stimu-
late cell growth.19 E5 also inhibits apoptosis, and maintains the
epithelial cell in continuous replication. Much of the specific
activities and function of E5 are poorly understood, however,
it appears that this gene is involved mainly in early events and
has an unknown role in the later phases of viral replication.
Although it has oncogenic activity, E5 expression is usually
absent in malignant cervical cells,20 and this has led to sugges-
tions that E5 activity is not critical for epithelial transformation.
E5 also binds to the vacuolar H + ATPase and inhibits its ability
to lower endosomal pH, potentially impacting vesicular traf-
ficking. Indeed, E5 expression is also associated with MHC-I
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downregulation, suggesting a role for E5 in viral escape from
cellular immunity.
E6. The HPV E6 oncoprotein is a 150 amino acid zinc-
binding protein that does not possess enzymatic activity. E6 con-
tains two zinc finger motifs, which possess four C-X-X-C motifs
necessary for E6 function.21 The E6 oncoprotein induces cellular
transformation, however, on its own, it has weak transforming
properties, but serves to amplify the transforming effect of HPV
E7 oncoprotein, which will be discussed later. E6 interacts pri-
marily with the p53 tumor suppressor protein,22-24 as well as with
other cell cycle regulators like Bak, Blk and cell adhesion pro-
teins. It is known that the E6 oncoprotein of low-risk HPV types
is less potentiating than that of high risk HPV types with respect
to cell transformation and immortalization activity.25
The E6AP is an E3 ubiquitin ligase that is responsible for
the targeted proteasomal degradation of cellular proteins. The
LXXLL domain on E6AP has been characterized as the binding
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Figure 4. Low-grade lesion with skip pattern.
site of E6 proteins of the alpha papillomaviruses. High risk HPV
E6 oncoprotein first binds to E6AP, before it is able to bind p53
to form a trimer. Within this trimeric complex, E6AP targets
p53 for disposal by proteosomes in the cervical epithelial cell
via ubiquitination.21 Under normal cell conditions, p53 exists in
low levels, tightly regulated by the E3 ubiquitin ligase activity of
Mdm2. The low level of p53 in normal epithelial cells is upregu-
lated via inhibition of Mdm2 when DNA damage occurs or when
abnormal cell proliferation signals are detected.22 In response to
the upregulation of p53 activity, several anti-proliferation path-
ways are activated. One of these is the activation of genes via the
action of novel proteins such as p300.22,26 The loss of p53, which
is a key regulator of uncontrolled cell proliferation, ensures that
the cervical epithelial cell is unable to exit the cell cycle. High
risk HPV E6 binds to the core region of p53, thereby inducing
p53 degradation, which occurs when the core region is bound.
This ability to degrade p53 is unique to high-risk HPV types, as
the low risk types do not bind to the core region. Also, high-risk
HPV E6 has been shown to bind p300, causing deacetylation of
300 Cancer Biology & Therapy Volume 11 Issue 3
p53 and preventing the activation of downstream pro-apoptotic
genes. Continued E6-E6AP binding leads to the targeting of the
Blk protein for breakdown, with the resultant effect of unchecked
cell proliferation.27
E6 also induces cell proliferation by targeting the expression
of apoptotic proteins other than p53. E6 binding leads to the
degradation of Bak, a Bcl-2 family pro-apoptotic protein that is
crucial in mitochondrial pore formation during initiation of the
intrinsic apoptotic cascade. Bak degradation prevents the release
of cytochrome c from the mitochondria, and prevents the down-
stream activation of caspases necessary for apoptosis.27,28 E6 of
high-risk HPV type has also been shown to disrupt the extrinsic
apoptotic pathway by binding to TNFR-1 (tumor necrosis factor
receptor), Fas, CD95 ligand and TNF related apoptosis induc-
ing ligand (TRAIL) receptors,28 thus inhibiting the downstream
release of caspase 8 and caspase 10, as well as inhibiting the for-
mation of the death inducing signaling complex (DISC).
As mentioned above, the cervical basal (epithelial) cells rest on
the basement membrane, glued to the extracellular matrix (ECM).
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Figure 5. Invasive cervical cancer in a 23 year old patient with HIV infection.
Cell signals from the ECM, which travel through the actin cyto-
skeleton of the basement membrane to the cell nucleus, play a role
in basal cell growth; once basal cells begin their ascent through
to the epithelial surface, the growth signals stop and epithelial
differentiation dominates. This transition is tightly regulated and
disruption of this process is one of the mechanisms by which E6
transforms cells. The anchoring of the basal cells to the ECM
involves paxillin and zyxin adhesion molecules. Their binding by
E6 leads to disruption of normal cell attachment and cell signal-
ing. In concert, the detachment of the epithelial cells from the
extracellular matrix, as well as the prevention of terminal dif-
ferentiation, serve to maintain an environment of continued viral
replication within continuously dividing epithelial cells.
The E6 protein of high-risk HPV types possess a PDZ-binding
motif that is critical for cell transformation through the binding
of cell proteins that have PDZ domains. In vitro studies have
identified these proteins such as hScribble and hDLG (tumor
suppressors), MUPP1 and PATJ (tight junction proteins).22,29
The proteosomal degradation of these proteins by E6 binding
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deregulates proliferation and disrupts the cell-cell junctions via
disruption of signals that regulate cell-cell contact. PDZ binding
appears to be an interaction that is necessary for carcinogenesis,
since E6 oncoproteins isolated from almost all invasive cervical
cancers possess this highly conserved PDZ binding motif.23,28,30
E6 in low risk HPV types have not been shown to possess this
PDZ binding motif.
Tissue culture studies have suggested a key mechanism by
which E6 immortalizes epithelial cells through the stimulation
of telomerase activity. High-risk HPV type E6 has been shown
to activate telomerase through E6AP binding to activate hTERT
(human telomerase reverse transcriptase). This process is also
dependent on the c-Myc oncogene, which acts on hTERT pro-
moter to increase its transcription. In addition, it appears that in
vivo, E6-E6AP binding increases hTERT activity through inter-
action with two isoforms of a gene repressor, NFX-1 (nuclear fac-
tor binds to the X1 box). The ubiquitination of NFX1-91 prevents
repression of the hTERT promoter, while binding to NFX1-123
leads to direct induction of hTERT promoter activity.22
 E7. E7 contains about 100 amino acids and, like E6, lacks
intrinsic enzymatic activity.31,32 E7 exerts its oncogenic effect
primarily by binding with retinoblastoma protein (pRb) and
the related pocket proteins, p107 and p130. By modulating E2F
transcriptional factors, these proteins regulate cell proliferation
by controlling entry at the G1/S phase of the cell cycle. Both
low risk and high risk HPV E7 bind to pRb, however, the low
risk HPV E7 has much lower affinity due to a single amino acid
substitution in the binding domain. E7 binding to pRb results
in proteosomal degradation of pRb and the unrestricted tran-
scriptional activity of E2F.31-33 Direct binding of E7 to E2F also
ensures transcriptional activity that maintains the epithelial cell
so they are always ready to enter S phase. E2F regulates the S
phase of the cell cycle by control of cyclins, specifically cyclins
A and E. Through the pRb inhibition and direct E2F induction
mentioned above, E7 drives cell proliferation with increased lev-
els of cyclins and also inhibits the activities of cyclin-dependent
kinase inhibitors (CKI), p21 and p27.33 E7 binding to pRb is also
linked to the expression of c-jun, which is important in control-
ling cell differentiation and growth. In effect, the inhibition of
pRb and c-jun ensure that the cell is maintained in a state of S
phase progression.34
Similarly, E7 binds to histone deacetylases (HDACs) with
the resultant effect of increasing E2F-driven transcription and
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S phase progression in epithelial cells. In addition to cell cycle
dysregulation, E7 mediates various structural changes in the
genomic structure of epithelial cells, notably extra chromosomal
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material, polar mitoses, anaphase bridging and aneuploidy. For
example, E7 uncouples centriole synthesis from cell cycle con-
trol, resulting in aberrant numbers of centrioles and chromosome
missegregation. The activation of cdk2 by E7 is thought to be
partly responsible for these changes,34 and in vitro experiments
have demonstrated that cdk2 inhibition is associated with a
decreased frequency of these chromosomal changes.
E6 and E7 Immune Modulation
In addition to the combined expression of E6 and E7 in high-risk
HPV types greatly enhancing cellular transformation, E6 and
E7 also modulate molecules involved in the innate and adaptive
immune responses, potentially enabling HPV to evade immune
surveillance, especially in the early stages of replication.
E6 inhibits the ability of interferon regulatory factor 3 to
induce the activation of interferon beta, thus abrogating this early
response of the innate immune system to viral infection. E7 also
binds to interferon regulating factor 1 to prevent activation of
interferons alpha and beta. Likewise, the inhibition of toll like
receptor 9 (TLR9) and subsequent failure of cytokine production
by the expression of E6 and E7 has been demonstrated in tissue
culture studies.29
Cervical cancer cells show an abnormal response to the action
of transforming growth factors (TGFs), which typically act to
prevent uncontrolled epithelial cell growth. Downregulation of
tumor necrosis factor (TNF)alpha expression, as well as resis-
tance to the effects of TNF alpha and beta, have been observed in
cervical cancer cells. While not entirely understood, it is thought
302 Cancer Biology & Therapy Volume 11 Issue 3
that E7 inhibition of CKIs p15, p21 and p27 is partly responsible
for the absence of effects of TGFbeta in infected epithelial cells.29
E6 modulates the contact between epithelial cells and antigen
presenting cells (Langerhans cells) in the epidermis by affecting
the cell surface protein, E-cadherin. Diminished Langerhans cell
numbers and reduced E-cadherin are recognized features of HPV
infection. Epithelial cells normally secrete cytokines to induce
chemotaxis, which leads to the repopulation of Langerhans cell
precursors.27 This chemotactic activity is mediated through the
macrophage inflammatory protein, MIP-3alpha. MIP-3alpha
expression is inhibited in cells that show evidence of E6 and E7
expression with the resultant impairment of antigen presentation.
In addition to the above E6 and E7-mediated immune effects,
the HPV replication cycle appears to confer certain survival
advantages that allow for HPV infections to persist for long peri-
ods in certain individuals. The intraepithelial sequestration of the
replication cycle allows for the absence of a blood borne phase.23
This absence of viremia allows viral replication to proceed some-
what covertly without triggering the immune system. The release
of viral particles from the terminal (apical or top layer) epithe-
lial cells, as well as non-lytic replication, avoids provocation of
anti-inflammatory molecules. Asymptomatic HPV infections are
the norm in immunocompetent women, even in those with high-
risk HPV infections, because most (younger) women clear their
infections within 12 months.35 This observation is obviously con-
founded by the fact that it is quite possible for the same patient
to acquire different HPV types going forward in time, such that
the contribution of one or an other HPV type to the clinical pic-
ture of persistence is hard to separate. The reason for persistence
and progression of lesions in a minority of patients is not well
understood. The average lag time from initial HPV infection to
carcinogenesis is 1020 years.35 The integration of viral DNA
into host cells is a typical event in carcinogenesis, but is thought
to occur sporadically. This suggests that there may be other, yet
unknown, environmental and genetic factors that influence the
propensity of HPV to remain latent, regress or progress with time.
Clinical Aspects of HPV Infection
Premalignant Cervical Disease
The cervix normally contains a central canal (endocervical canal),
which is the caudad continuation of the endometrial canal of the
uterus. Columnar cells and squamous cells line the proximal and
distal endocervical canal, respectively.36 The squamo-columnar
cellular junction is an active transition zone known as the trans-
formation zone (TZ), which is the site of origin of the majority
of cervical dysplastic (premalignant) lesions and carcinomatous
lesions. In premenarcheal and postmenopausal women, hor-
monal and physical changes cause the transformation zone to
recede into the cervical canal, hence making it sometimes dif-
ficult to visualize on vaginal examination. In reproductive age
women, the acid environment of the vagina leads to destruction
of the columnar cells of the TZ, and induces squamous metapla-
sia in the TZ. This metaplastic change (clinically considered as
benign) occurs when the reserve cells underlying the columnar
epithelium differentiate into a squamous type epithelium. HPV
 Table 2. Rates of regression and progression of cervical dysplasia
lesions36
Regress % Persist % Progress %
CIN 1/2 57 27 15
CIN 3 8 69 22
infection can induce change from normal to dysplastic cellular
architecture (clinically considered as abnormal) in the transfor-
mation zone and surrounding cervical portio.
The old terminology for cervical dysplasia was Cervical
Intra-epithelial Neoplasia or CIN, which has since changed
to Squamous Intraepithelial Lesion (SIL) following the 2001
revision of the Bethesda Classification of cervical dysplasia.36
Cervical cancer screening involves the initial step of accessing
squamous cells in the transformation zone, for cytological analy-
sis (Papanicolau smear or Pap smear). Cervical cytology reports
classify dysplastic lesions as low grade or high-grade squamous
intraepithelial lesions (LSIL, HSIL respectively), as well as atypi-
cal squamous cellsunknown significance, or cannot exclude
high-grade (ASC-US, ASC-H respectively). Cervical glandular
cell abnormalities may also be reported, but will not be covered in
this discussion. If indicated, the next step following a Pap smear
is the assessment of the cervix using a microscope (colposcopy).
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Colposcopy usually facilitates the obtaining of biopsies from
the cervical lip and endocervical canal. Histological reports of
colposcopic biopsy specimens are reported as CIN 1, 2 or 3. In
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2001, LSIL and HSIL were introduced in the Bethesda revision
colposcopic biopsy descriptions; however, CIN is still used in
many biopsy reports.36
LSIL typically involves the basal one-third of the cervical
epithelial layer (above the basement membrane and stroma),
and occurs as a result of HPV cytopathic effect of a productive
lesion, i.e., the complete virus life cycle. LSIL lesions display
a pathognomic change known as koilocytosis.37 Koilocytosis
refers to the presence of nuclear atypia in combination with
cytoplasmic cavitations, which gives a vacuolated appearance
to the cells. The cytoplasm takes on a granulated and coarse
appearance under the microscope. In addition, abnormali-
ties in the chromosomal structures involved in mitosis create
a multinucleated appearance of the epithelial cells. Presence of
immature basaloid cells in the upper third of the cervical epithe-
lial layer characterizes HSIL. Affected cells show cytoplasmic
granulation, nuclear crowding, increased mitotic activity, cel-
lular crowding (loss of polarity), as well as variations in the size
of their nuclei (anisonucleosis).38 There may also be an increase
in the nuclear to cytoplasmic size ratio due to shrinkage of the
cytoplasm. Under the old terminology, HSIL corresponds to
CIN 2 and CIN 3. The most severe HSIL change (formerly
termed carcinoma-in situ, CIS) involves the whole thickness of
the cervical epithelium, however, an important distinguishing
feature from invasive carcinoma is the absence of disruption of
the basal membrane of the epithelial layer and lack of invasion
into the cervical stroma.39 ASCUS lesions denote a group of dys-
plastic lesions that are not clearly LSIL or HSIL; in other words,
lesions that the pathologists do not feel comfortable describing
www.landesbioscience.com Cancer Biology & Therapy 303
Table 3. HPV genes and their functions
HPV gene Function
Viral replication
E1, E2 Autoregulation of E1 and E2 function by E2
Repression of E6 and E7 expression by E2
Inhibition of apoptosis
E5
? Immune dysregulation
p53 degradation
Inhibition of apoptosis
E6 Cell cycle progression
Cell transformation
Immune dysregulation
pRb inhibition
Cell cycle progression
E7
Cell transformation
Immune dysregulation
E4 ? Facilitates release of mature viral particles
Synthesis of viral capsid
L1, L2
Antigenicity
as mild, but which may not show overt features of a high-grade
process. From a clinical perspective, one of the most challenging
dilemmas has been the issue of avoiding overtreatment of inde-
terminate and low-grade lesions, while trying to devise clinical
thresholds to capture lesions that have a reasonably high chance
of progressing to cancer. Table 2 shows regression, persistence
and progression rates for dysplastic lesions. Please see Table 3.
The management of these lesions is discussed below.
Clinical Aspects of HPV Infection
Management Strategies
Cervical Pap smear screening in women in the US is instructed
to commence three years after initiation of sexual activity, and
no later than age 21 years. Of the 50 million cervical Pap smears
done annually in the US, about 34% are reported as LSIL,
0.6% as HSIL and 46% as ASCUS.35 In recent years, high-risk
type HPV testing has been used to guide the management of
abnormal cervical Pap smears. The ASCUS/LSIL Triage Study
(ALTS), carried out from 199598, was reported as a random-
ized trial of 3,488 women with ASCUS Pap smears and 1,572
women with LSIL Pap smears, using HPV testing to determine
colposcopy referral and setting HSIL as the threshold lesion for
colposcopy referral (study endpoint).40,41 Women were random-
ized to three groups; immediate colposcopy (IC), HPV testing
and conservative management (CM) groups.
In the LSIL part of the study (n 1,572), a breakdown of the
enrollment Pap smears on re-examinations were as follows; nega-
tive 18.7%, LSIL 45%, ASCUS 23.2%, HSIL (CIN 2) 11.3%
and HSIL (CIN 3) 1.4%. Using the triage strategies of IC,
HPV test and CM, the sensitivities for detecting CIN 3 over
two years follow up (study endpoint) were 55.9, 65.9 and 48.4%
respectively (p 0.16). Based on these detection sensitivities, the
proportion of patients subsequently referred to colposcopy were
100% (as expected for immediate colposcopy strategy), 85.3%
and 18.8% respectively (p < 0.001). Also, the finding of LSIL was
associated with a 25% risk of having CIN 3 at two years follow
up evaluation.
In the ASCUS part of the study, (n 3,488), a breakdown of the
enrollment Pap smears on re-examinations were as follows; nega-
tive 41.9%, LSIL 18.2%, ASCUS 32.4%, HSIL (CIN 2) 5.9%
and HSIL (CIN 3) 1.1%. Using immediate colposcopy, HPV
testing and conservative management, the detection sensitivities
for CIN 3 lesions were 53.6, 72.3 and 54.6% respectively (p <
0.01). The respective colposcopy referral rates were 100, 55.6 and
12.3% (p < 0.001). HPV testing had the highest detection rate.
ASCUS pap smears were associated with a 15% rate of persistent
CIN 3 lesions over two years of follow up.
A re-analysis of the ALTS trial data (n 5,060),42 showed that
the cumulative incidence of high-grade lesions (CIN 2, 3) was
similar among the three study arms (IC 10.9%, HPV testing
10.9% and CM 10.3%). Forty percent of CIN 2 lesions were
noted to regress over the two year follow-up period, however,
CIN 2 lesions that tested positive for HPV 16 were less likely
to regress. The significance of these results lies in the fact that
HSIL (CIN 2, 3), regardless of HPV status, is a trigger for col-
poscopy referral, and thus, the issue of overtreatment of HPV
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negative lesions is a potential problem. Furthermore, all the
cases of HSIL (CIN 2, 3) were diagnosed based on ASC-US
and LSIL referrals into the ALTS study. ASCUS and LSIL are
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the most prevalent Pap smear abnormalities in the screening
population, and HPV 16 is the most prevalent high-risk type.
Consequently, CIN 2, 3 could represent a potentially large pro-
portion of abnormal cervical pathology that would need to be
managed clinically.
Current Clinical Management Recommendations
It is now established that HPV is highly prevalent, and the high-
risk oncogenic types, notably HPV 16, are associated with more
severe dysplastic changes in the cervix. Nonetheless, immuno-
competent individuals normally clear most HPV infections
and the majority of low-grade lesions regress spontaneously in
younger women; likelihood of regression to normal is 60% with
CIN 1 lesions, and 40% with CIN 2.9,11,43,44 While cervical can-
cer is rare among women less than 25 years old, this age group
is known to present with cytological findings that may provoke
tests and interventions that are unnecessary.
In general, among patients with LSIL on Pap smear, the high
frequency of positive HPV tests resulted in an inefficiently high
referral rate to colposcopy.41 Furthermore, the use of colposcopic
evaluation in LSIL appears to increase cost without necessar-
ily providing a benefit in increasing the detection of high-grade
lesions. In contrast, HPV testing among women with ASC-US
cervical Pap smears is useful in making clinical decisions to refer
for further colposcopic evaluation of the cervix. HPV testing had
an acceptable detection rate for high-grade lesions while avoiding
the unnecessary costs of referring every patient for colposcopy.40
Conservative management of ASC-US lesions has a low rate of
304 Cancer Biology & Therapy Volume 11 Issue 3
colposcopy referrals, and this is troublesome for a low detection
rate and higher chance of missing high-grade persistent lesions.
Based in part on clinical data reviewed above,40-42 the
American College of Obstetricians and Gynecologists (ACOG),
and the American Society for Colposcopy and Cervical Pathology
(ASCCP) have endorsed revisions to cervical Pap smear screening
and management as follows:
The time interval for Pap smear testing of women considered
to be at low risk (using risk factor screening and prior Pap smear
results) has been extended from one- to two-yearly. Women over
the age of 30 years who have negative HPV tests, preceded by at
least two consecutive negative Pap smears can be screened once
every three years. Among women in this group, the 10 year risk of
CIN 2 is 25%, and of CIN 3 is <2%. Women who are between
6570 years old, with a low risk profile, may discontinue Pap
smear screening altogether.
Low-grade dysplasia is now managed conservatively by obser-
vation (in adolescent girls) and surveillance to confirm that the
lesion has regressed. In older reproductive age women, LSIL is
managed by performing colposcopy (27% have CIN 2 on col-
poscopic evaluation). HPV testing is usually not done, and the
results of prior HPV tests do not influence the management.
ASC-US (atypical, unknown significance) Pap smears, the
most common category, are now triaged by performing high-
risk type HPV testing to determine the need for colposcopic
examination and biopsy in patients who test positive for HPV.
Women who test negative for HPV are managed conservatively
with repeat Pap smears. In contrast, ASC-H (atypical, suspect
high grade) is managed by colposcopic examination and biopsy
to exclude high-grade dysplastic lesions in the cervix or endocer-
vical canal (6785% are HPV positive, and have a significantly
higher chance of harboring CIN 2, 3 on colposcopic evaluation).
High-grade dysplastic lesions on Pap smears (HSIL) are evalu-
ated with immediate colposcopic examination and biopsy (data
has shown that 5366% of women with HSIL harbor CIN 2, 3
on colposcopic evaluation). HPV testing for high-risk HPV types
is also useful in these patients. In women past their reproduc-
tive years, HSIL can be managed by surgical excision such as
cold knife conization or loop electrosurgical excision. In younger
women, excisional procedures are avoided because of the poten-
tial complications of cervical conization such as infertility, cervi-
cal incompetence and premature labor and delivery. Discussion
of the management of post-colposcopy histological findings is
beyond the scope of this article. Please see Figures 15.
Human Papillomavirus Vaccines
In June 2006, the United States Food and Drug Administration
(FDA) approved the prophylactic quadrivalent HPV vaccine
(GARDASIL, Merck and Co., Inc.), for use in women aged
926 years old.45 Subsequently, in October 2009, the prophylac-
tic bivalent HPV vaccine (CERVARIX, GlaxoSmithKline) was
licensed for use in women aged 1025 years old.46 The quad-
rivalent vaccine is composed of recombinant L1 protein-based
viral like particles from HPV 6, 11, 16 and 18. Phase II and
phase III studies have shown efficacy of 100% in preventing
cervical dysplastic lesions in women aged 1626 years, who
were not infected by any of the vaccine HPV types.47 Efficacy
in women with simultaneous vaccine type HPV DNA positivity
and seropositivity was about 25%. The most common adverse
effects were related to injection site pain (84 vs. 48.6% in treat-
ment and placebo groups respectively). It is expected that the
vaccines will achieve a lifetime risk reduction of 2070% for
cervical cancer, in patients aged 12 years. The vaccine is typi-
cally given in three doses of 0.5 ml at 0, 2 and 6 months. The
bivalent vaccine contains recombinant L1 protein from HPV 16
and 18, and phase III study of 18, 644 females followed for 35
months showed efficacy of up to 93% in prevention of CIN 2
lesions due to HPV 16 and 18.46 Similar to the quadrivalent
vaccine, there was a higher rate of complications at the injection
site. The dosage schedule is 0.5 ml at 0, 12, and 6 months for
a total of three doses. New approaches to vaccine development
may provide more options for controlling HPV acquisition and
transmission in the future. The use of recombinant L2-based
prophylactic vaccines (Roden R, unpublished data), as well as
the development of drug targets against early protein (E6 and
E7) activity are a few examples.
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Conclusion
The study of HPV has provided valuable information on the
mechanisms by which tumor viruses initiate carcinogenesis
and the regulation of cell cycle in human cells. Cervical cancer
as a disease model presents unique opportunities in finding a
cure, because it is caused by a pathogen with well character-
ized biological mechanisms and life cycle. The use of cytologic
and HPV screening and ablation of premalignant disease has
proven very successful at reducing cancer rates in developed
nations. The recent introduction of prophylactic HPV vac-
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ease burden in the developing parts of the world, it is evident
that population based measures offer the best hope of limiting
mortality worldwide. The development of therapeutic cervical
cancer vaccines and/or virus-targeted drug therapies is eagerly
awaited.
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Dr. Ibeanu wishes to acknowledge Dr. Richard B. Roden for
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