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Human papillomavirus and

Review
Future Microbiology
cervical cancer: biomarkers for
improved prevention efforts
Vikrant V Sahasrabuddhe1, Patricia Luhn1 & Nicolas Wentzensen†1
1
Division of Cancer Epidemiology & Genetics, National Cancer Institute, National Institutes of Health, 6120
Executive Blvd EPS 5024, Rockville MD 20852, USA
†
Author for correspondence: Tel.: +1 301 435 3975 n Fax: +1 301 402 0916 n wentzenn@mail.nih.gov

While organized screening programs in industrialized countries have significantly

reduced cervical cancer incidence, cytology-based screening has several
limitations. Equivocal or mildly abnormal Pap tests require costly retesting or
diagnostic work-up by colposcopy and biopsy. In low-resource countries, it has
been difficult to establish and sustain cytology-based programs. Advances in
understanding human papillomavirus biology and the natural history of human
papillomavirus-related precancers and cancers have led to the discovery of a
range of novel biomarkers in the past decade. In this article, we will discuss the
potential role of new biomarkers for primary screening, triage and diagnosis in
high-resource countries and their promise for prevention efforts in resource
constrained settings.

Cervical cancer: incidence & burden and effective methods of treatment of cervical
Invasive cervical cancer (ICC) is a significant precancer. Yet concerns about the substantial
cause of cancer-related morbidity and mortality cost burden associated with screening, lim-
among women worldwide, with substantial geo- ited accuracy of cytology and complications of
graphic variation [1] . Many industrialized coun- unnecessary treatment have prompted research
tries have achieved significant successes in reduc- and development of more efficient approaches
ing ICC burden over the past six decades, and for cervical cancer prevention. Over the past two
with annual incidence rates between 4 and 14 decades, substantial improvements in under-
per 100,000, ICC no longer ranks even among standing the natural history of HPV-associated
the top ten cancers in these settings. The low cervical carcinogenesis as well as advancements
incidence is achieved through substantial health- in molecular technologies have led to the avail-
care investments for screening programs and ability of novel screening tests that provide
diagnostic workup in these countries. On the alternatives or adjunctive methods to cytology.
other hand, cervical cancer is the leading can- Prominent among these are the HPV-DNA
cer among women in many resource-constrained based screening assays, already widely used as
settings of the developing world, where incidence adjunctive methods for primary screening and
and mortality rates are about five- to six-times for triage of equivocal cytology. At the same
higher [1] . Rates are highest in sub-Saharan time, HPV vaccines have been developed and
Africa, South-Central Asia and parts of South introduced that have high efficacy in prevent-
America, where ICC represents from a sixth up ing HPV infections when administered in HPV-
to a fifth of all cancers among women [1] . naive populations. There is unanimous agree-
Persistent infections with carcinogenic human ment that screening efforts have to continue,
papillomavirus (HPV) genotypes have long been and screening algorithms have to be made more
established as the necessary, but not sufficient, efficient, since it will take years to decades to see
cause of ICC [2,3] . Organized prevention pro- an effect of HPV vaccination on reduction in
grams in industrialized settings have relied on cancer incidence. In addition, newer screening
early detection of HPV-associated dysplastic approaches are needed to anticipate continuous
changes in exfoliated cervical cells (‘Pap smear’) changes in disease prevalence in populations Keywords
that reflect underlying precancerous lesions [4] . with increasing vaccination coverage. n accuracy n biomarkers
Cervical cancer screening has been a success n cervical cancer n HPV
owing to the long period, typically extending Biomarker principles n prevention n risk prediction
n screening
over many years, carcinogenic HPV infections In this article, we discuss the current evidence
take to progress to precancerous lesions (cervi- and opportunities for improvement of cervi- part of
cal intraepithelial neoplasia, grade 3 or CIN3) cal cancer screening through the use of novel
and ICC, and the availability of relatively safe biomarkers. In many countries, Pap cytology is

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 Review Sahasrabuddhe, Luhn & Wentzensen

still the primary screening test, either alone or in n The upper regulatory region which functions
conjunction with HPV testing (predominantly as a transcription and replication control
in the USA) [5–8] . In some European countries, region;
a switch to primary HPV screening followed by n An ‘early’ region encoding proteins (E1, E2,
cytology triage has now been recommended [9] .
E4, E5, E6, E7) for replication, regulation and
New biomarkers may have potential use in pri-
modification of the host cytoplasm and
mary screening, as triage tests for primary cytol-
nucleus;
ogy screening, and as triage tests for primary
HPV screening. For any biomarker to be use- n A ‘late’ region encoding the viral capsid
ful, the test result has to influence clinical man- p­roteins (L1, L2).
agement. Management options include direct
referral for treatment, referral to colposcopy to The prominent areas of research focused on
confirm precancer histologically, increased sur- biomarker discovery and validation are concep-
veillance through more intensive screening or tually based on events in the HPV life cycle and
release to routine screening. The management natural history of HPV-dependent cervical car-
options should be chosen based on an individ- cinogenesis. While the phylogenetic taxonomy
ual’s risk of precancer and cancer, indicated by and classification of papillomaviruses continues
screening test results and other risk indicators to be refined, 13 HPV genotypes (HPV types
such as age [10,11] . 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,
When assessing screening options, it is impor- 68) are considered carcinogenic while some oth-
tant to consider physical and financial harm asso- ers (HPV types 26, 53, 66, 67, 70, 73, 82) are
ciated with unnecessary tests and procedures. considered possibly carcinogenic in humans [15] .
False-positive test results may cause anxiety, The molecular mechanisms of how HPV causes
lead to overtreatment of women, increase risks cancer have been extensively studied. Two viral
of obstetric complications, and thus increase the oncoproteins, E6 and E7, interfere with key cel-
downstream costs of a screening program. The lular pathways that control cell proliferation and
goal of cervical cancer screening programs is to apoptosis. Specifically, E7 disrupts pRb from its
prevent cancer, not to treat cervical intraepithe- binding to E2F and triggers uncontrolled cell
lial neoplasia. Currently, a treatment threshold cycling. E6 interferes with p53 and abrogates
of CIN2 or worse lesions is widely used, despite apoptosis, which would normally occur in cells
the fact that a large percentage of CIN2 lesions with uncontrolled cell proliferation. E6 and
spontaneously regress [12] . Furthermore, there E7 induce substantial chromosomal instabil-
is increasing evidence that even CIN3 is a het- ity in transformed cells, even at precancerous
erogeneous group; only about 30–50% of large stages [15,16] . While biomarker discovery con-
CIN3s are estimated to invade to cancer over tinues in multiple directions, current biomarker
a long time period [13,14] . An important area of candidates can be broadly categorized into two
cervical cancer biomarker research focuses on groups, viral or cellular markers (Figure 1) . The
the identification of markers for cervical lesions biomarker research pipeline extends from dis-
that likely progress to cancer. It is important to covery (in vitro/preclinical studies) to early stage
note that risk thresholds and available resources validation, and then to validation in randomized
can vary substantially between populations and clinical trials [17] . A tabular representation of
may lead to different screening recommenda- the state of availability and status of regulatory
tions based on the optimal trade-offs between approval of commercially marketed biomarkers
benefits and harms. is presented in Table 1.
In the first part of this article, we briefly sum-
marize the evidence on biomarkers that have The limitations of cervical cytology
been widely evaluated and are already in lim- Cytology was introduced in the early 1950s as
ited use in clinical practice. In the second part, a primary screening method as part of annual
we discuss biomarkers in discovery and valida- preventive examinations, even though it was
tion phases that have the promise to improve never subject to evaluation for effectiveness in
s­creening in the future. randomized trials. The declining incidence rates
in settings in Northern America, Europe, and
HPV life cycle & natural history & the Australia have provided widely accepted proof
basis for biomarker selection of the effectiveness of cytology-based screen-
The HPV genome consists of a circular double- ing [1,18] . Screening with cytology has become
stranded, 8000 bp long DNA with three regions: a well-established component of standard

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 Human papillomavirus & cervical cancer: biomarkers for improved prevention efforts Review

Chromosomal instability:
3q, 5p

Cellular
Proliferation: MCM2, Top2a, ki-67

p16ink4a

Host and viral methylation

HPV integration

Viral
HPV oncogene mRNA

HPV DNA detection

Infection Progression Invasion

Invasive
Normal HPV Precancer cervical
infected cancer
Clearance Regression

Figure 1. Human papillomavirus natural history and cellular and viral biomarkers used in
cervical cancer screening. HPV infection happens shortly after sexual initiation. Most infections
clear spontaneously, but a few carcinogenic HPV infections may persist and initiate oncogenic
changes in epithelial cells at the cervical transformation zone. In a small fraction of cases, these
persistent abnormalities may progress to invasive cervical cancer in the absence of early detection and
treatment. Viral and cellular biomarkers indicating key steps of the functional progression model
(HPV infection, precancer and invasive cancer) have been discovered, with some currently in early
discovery stages, while others have already been commercialized.
HPV: Human papillomavirus.

preventative care in most industrialized settings CIN3. Since ASC-US and LSIL represent signif-
[19,20] . For example, more than 80% of women icant proportions of cytological results, further
surveyed in a US study reported receiving Pap workup is required to make management deci-
smears in the past 3 years [18] . In fact, over half sions [29] . It is expected that cytological screening
of incident cases of ICC in the US continue to will be especially challenged in HPV-vaccinated
occur in women who have never or rarely been populations, as the reduction of CIN2+ preva-
screened [21] . Although the clinical sensitivity lence will be much higher than the reduction of
of a single Pap smear is quite modest (60–70%) low-grade abnormalities, further decreasing the
[22,23] , the success of cytology-based screening signal-to-noise ratio of cytology testing [31,32] .
is achieved by frequently repeated Pap testing, Finally, most promising efforts to curb rising
causing substantial cost burdens on the health- healthcare costs rely on prolonging screening
care system [24,25] . Multiple efforts have focused intervals through improvements in negative pre-
on improving the accuracy and cost effective- dictive value of the screening test, a weakness of
ness of cytology-based screening protocols [26] . low-sensitivity cytology screening [32] .
The introduction of liquid-based cytology has
decreased the proportion of inadequate slides, The role of HPV DNA testing in cervical
and has permitted reflex testing for other molec- cancer screening
ular markers [27,28] . Yet false-negative rates asso- Testing for HPV DNA, the necessary cause of
ciated with cytology continue to be substantial, virtually all ICC, provides a biologically salient
primarily since cytological detection still relies approach for screening [33,34] . The detection of
on visual identification and subjective interpre- HPV in cervical scrapings was one of the first,
tation of morphologic changes induced by car- and to date is the most widely evaluated, alterna-
cinogenic HPV [27] . About 10–15% of women tive to cytology [26,35] . Current HPV detection
with equivocal (atypical squamous cell of unde- technologies are focused on hybridization with
termined significance [ASC-US]) [29] and mildly signal amplification of HPV DNA (e.g., Digene
abnormal (low grade squamous intraepithelial Hybrid Capture ® 2 (hc2) (by Qiagen) [36] ;
lesions [LSIL]) [30] results have an underlying Cervista® HPV HR (by Hologic) [37]) or genomic

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 Review Sahasrabuddhe, Luhn & Wentzensen

Table 1. Major classes of biomarkers being developed and validated for use in cervical cancer
prevention research.
Type of Test format Application Quality of evidence/ Manufacturers and test names
biomarker regulatory approval
Viral markers
Detection of Signal amplification (e.g., Primary screening Large population-based Qiagen: Digene hc2, careHPV™,
carcinogenic Digene Hybrid Capture-2) Triage of equivocal studies and randomized QIAensemble™†
HPV DNA Target genome cytology trials Roche: Amplicor®, Cobas® 4800†,
(and HPV amplification by PCR (e.g., Many tests licensed for Linear Array®‡
genotyping) Amplicor®, Linear Array®) use in the USA and Cervista® HPV HR†
Europe, many in final CLART® HPV2‡
regulatory stages Autogenomics: Infiniti® HR-HPV QUAD‡
BioRad: HR-HPV Dx PCR
Innogenetics: InnoLiPA™‡
Multimetrix: Multiplex HPV
Genotyping Kit‡
Greiner: Papillocheck® HPV-Screening‡
Abbott: RealTime HR HPV®†
Not commercialized: GP 5+/6+ EIA‡
Detection of Nucleic acid sequence- Adjunct to primary Multiple clinical studies GenProbe: Aptima®
E6/E7 mRNA based amplification HPV-based published Norchip: PreTect® Proofer‡
Transcription-mediated screening Large population-based BioMerieux: NucliSENS EasyQ® HPV‡
amplification Triage of equivocal studies underway IncellDx: HPV OncoTect®‡
In situ hybridization or mildly abnormal
cytology
Detection of Immunostaining of Adjunct to primary Some clinical studies Cytoimmun: Cytoactiv®
HPV protein histology and cytology HPV-based published ArborVita: AVantage™ HPV E6
slides (L1) screening
ELISA (E6) Triage of equivocal
or mildly abnormal
cytology
Cellular markers
p16ink4a Immunostaining of Primary screening Multiple clinical studies mtm Laboratories: CINtec® and CINtec®
(also with histology and cytology Triage of equivocal published PLUS
addition of slides or mildly abnormal Large population-based
Ki-67) ELISA cytology studies underway
MCM2 and Immunostaining of Primary screening Some clinical studies Becton Dickinson: ProEx™C
TOP2A histology and cytology Triage of equivocal published
slides or mildly abnormal
cytology
Results include partial HPV genotyping.
†

Genotyping assay.
‡

HPV: Human papillomavirus; MCM2: Minichromosome maintenance protein 2; TOP2A: Topoisomerase IIA.

amplification using PCR (e.g., Amplicor® HPV that HPV DNA testing has a higher sensitiv-
Test and Cobas® HPV Test (by Roche) [38,39]), ity in comparison with cytology for detection
with most results reported as aggregate presence of CIN3 [26,47,48] . Yet its utility is constrained
or absence of carcinogenic HPV types. by its limitation of lower specificity than cytol-
The primary benefit of using HPV testing is ogy, since the majority of HPV infections are
the high sensitivity and high negative predictive transient and would not progress to cervical
value, since the absence of carcinogenic HPV dysplasia [49,50] . The high prevalence of benign
indicates an extremely low risk of CIN3/ICC and self-limiting HPV infections, and the low
for 5–10 years, thereby allowing for safe pro- prevalence of cervical cancer precursors (let
longing of screening intervals [32] . The role of alone ICC), in the second and third decades of
HPV DNA testing as a solitary primary screen- life further limit the use of HPV DNA testing
ing test (to replace cytology) or as an adjunct for these age groups. Hence, the use HPV DNA
to cytological screening has been evaluated in testing in primary screening is currently primar-
large randomized trials over the past decade ily focused on women 30 years or older [9] . At
[40–46] . Results show overwhelming evidence any age, however, a single negative HPV DNA

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 Human papillomavirus & cervical cancer: biomarkers for improved prevention efforts Review

test indicates a very low risk of precancer over prevention research has been on development
the next 5–10 years and allows clinicians to and validation of new disease-specific biomark-
extend screening intervals safely [51] . ers of HPV-associated transformation [63–66] .
Since the FDA approval of Digene hc2 as a test The underlying biological basis and utility of
for triage of ASC-US cytology in 2000, its use some prominent biomarkers is discussed below
has increased steadily in the USA [29,52] . In the and their functional relevance in relation with
ALTS trial, it was found that while HPV test- various stages of cervical carcinogenesis is
ing was deemed to have utility in distinguishing s­chematically presented in Figure 1.
women with ASC-US who were at risk for pre-
cancer, it was limited in its discriminating capac- E6/E7 mRNA detection
ity for mildly abnormal (LSIL) cytology given The progression from a transient to a transform-
the high background prevalence of carcinogenic ing HPV infection is characterized by a strong
HPV in this population [53] . The availability of increase of HPV E6/E7 mRNA and protein
genotype-specific information for HPV could expression [67] . Multiple studies have evaluated
potentially provide additional risk stratification the role of detection of mRNA transcripts in
in HPV-positive women. This may be of particu- cervical scrapings to identify cervical precancers
lar relevance in the detection of HPV types 16 [68–76] . At least two commercial platforms are
and 18, since HPV 16-associated lesions are more currently available: PreTect® Proofer (Norchip
likely to be persistent and have higher carcino- [marketed as NucliSENS EasyQ® by BioMerieux
genicity than other HPV types [54,55] , and since in some European markets]) and APTIMA®
HPV 18 is more associated with lesions within (GenProbe) (Table 1) . In a recent meta-ana­lysis by
the endocervical canal that are frequently missed Burger and colleagues [77] , 11 studies that evalu-
by cytology [56] . Indeed, some newer HPV DNA ated HPV E6/E7-based mRNA detection against
detection assays are able to provide type-specific HPV DNA testing for detection of CIN2+ refer-
information for HPV 16/18 [39,57–61] (Table 1) . A ence standard were summarized. Given the con-
typical application is HPV16/18 genotyping siderable heterogeneity, pooling of data was not
in HPV-positive, cytology-negative women. possible. A ‘best evidence synthesis’ for E6/E7
Positivity for HPV16/18 may warrant earlier mRNA HPV testing accuracy was provided, that
referral to colposcopy because of the higher risk reflected a sensitivity ranging between 0.41 to
associated with these types. However, it remains 0.86 for the PreTect Proofer/NucliSENS Easy
to be determined in clinical studies and cost- Q assays while a higher range – from 0.90 to
effectiveness analyses whether HPV genotyping 0.95 – for the APTIMA assay. The specificity
provides sufficient risk stratification in a screen- ranged from 0.63 to 0.97 and from 0.42 to 0.61
ing population. for the PreTect Proofer/NucliSENS EasyQ and
In the USA, cytology and HPV DNA co- APTIMA assays, respectively. The considerable
testing are being widely used. In Canada and difference in sensitivity (and specificity) between
many European settings, a strategy with primary PreTect Proofer/EasyQ and APTIMA may in
screening by HPV testing followed by cytology part be explained by the difference in type cov-
triage of HPV DNA positives (‘sequential’ or erage: The former tests detect only five types
‘two-stage’ testing) was proposed [32] and has (HPV16, 18, 31, 33, 45), while the latter covers
been evaluated in multiple randomized clinical 14 types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52,
trials [62] . This strategy takes advantage of the 56, 58, 59, 66, 68).
high negative predictive value of HPV DNA test-
ing and maximizes sensitivity, while reserving p16ink4a
cytology for those who have higher likelihood The biomarker most widely evaluated is p16ink4a,
of dysplastic lesions. The reliance on cytology, a cyclin-dependent kinase inhibitor that is mark-
with subjective interpretation and substantial edly overexpressed in cancerous and precancer-
inter-observer variability, along with potential ous cervical tissue. p16ink4a is a cellular correlate
for sampling/collection errors, however, remains of the increased expression of the viral oncopro-
a challenge. tein E7 that disrupts a key cell cycle regulator,
pRb, in transforming HPV infections. The dis-
Novel biomarkers in cervical turbance of the Rb pathway leads to a compensa-
cancer prevention tory overexpression of p16ink4a through a negative
Given limitations in use of both cytology and feedback loop [64] . The resultant overexpression
HPV DNA based approaches as standalone and cellular accumulation of p16ink4a is a spe-
tests for screening, the focus of cervical cancer cific marker of cervical precancerous lesions and

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 Review Sahasrabuddhe, Luhn & Wentzensen

can be measured through immunocytochemi- PLUS) has been introduced that is supposed
cal staining of histology and cytology slides and to substantially simplify and standardize the
using ELISA assays [78] . evaluation of stained slides [80,84] .
A commercially available CE-marked assay
(CINtec®, mtm Laboratories) has been widely Markers of aberrant S-phase induction
validated. Liquid-based cytology systems such The cell cycle activation mediated by HPV
as ThinPrep®, SurePath™, CYTO-screen sys- oncogenes in transforming infections is char-
tem® and others have been used in these studies. acterized by aberrant S-phase induction. An
p16ink4a has been evaluated as a standalone test assay detecting two proteins indicating aberrant
and as an adjunct to cytology [79–84] or HPV S-phase induction, topoisomerase IIA (TOP2A)
testing [80,85,86] . The role of p16ink4a based detec- and minichromosome maintenance protein 2
tion in screening and triage has been reviewed (MCM2) is commercially available (ProEx™ C
in previous articles [63,87] . These reviews noted by Becton Dickinson) [88] . Few clinical stud-
substantial heterogeneity in methods used ies with limited sample size have shown that it
for defining p16ink4a positivity in the cytology has a sensitivity ranging between 0.67 and 0.99
application, including quantitative and mor- and specificity ranging between 0.61 and 0.85
phologic approaches. The sensitivity has ranged (Figure 2) [89–94] .
between 0.59 and 0.96 and the specificity has
ranged between 0.41 and 0.96 for the detection Other biomarkers undergoing
of CIN2+ lesions in clinical studies, reflect- clinical validation
ing the heterogeneity in test interpretation and Other cellular makers such as CK13 and
analyzed populations (Figure 2). Recently, a dual CK14 [95] , MCM5 and CDC6 [96] , Survivin [97]
immunostain of p16ink4a with Ki-67 (CINtec® and CEA [98] have also been evaluated in various

TOP2A–MCM2
100 (ProEx™C) E6/E7 mRNA
(APTIMA®)

p16ink4a
(CINtec®)
75

Combined Sensitivity (%): Specificity (%):

number median median
Sensitivity (%)

of samples (Low–high range) (Low–high range)

50 evaluated
p16ink4a
4733 88 (59–96) 63 (41–96)
(CINtec®)
E6/E7 mRNA E6/E7 mRNA
3509 93 (90–95) 51 (42–61)
(PreTect® HPV Proofer) (APTIMA®)
25 E6/E7 mRNA 5003 76 (63–96)
77 (41–86)
(PreTect® HPV Proofer)
TOP2A MCM2 1824 86 (67–99) 75 (61–85)
(ProEx™C)

0
100 75 50 25 0
Specificity (%)

Figure 2. Graphical representation of the range of estimates of sensitivity and specificity of

studies evaluating commercially available biomarkers at the cervical intraepithelial
neoplasia grade 2+ threshold. The sensitivity ranges are plotted along the y-axis and specificity
along the x-axis. The median values of the range of estimates of sensitivity and specificity are used as
the points of convergence of the sensitivity and specificity range lines. The circles around each graph
reflect the combined total number of samples used in the studies that were summarized. This
graphical representation does not weigh the studies by sample size, and the median value does not
reflect a computed summary/pooled measure. The purpose of this graph is to visualize the wide
range of performance estimates reported in studies evaluating these biomarkers. The heterogeneity is
related to various factors, including but not limited to differences in targeted populations, differences
in clinical end points and heterogeneity of biomarker performance.

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 Human papillomavirus & cervical cancer: biomarkers for improved prevention efforts Review

stages of development. Most are marked by non- efforts are also being undertaken to evaluate
uniformity in determination of end points and biomarker assays using noninvasive and user-
limited sample sizes. Other viral markers such operated screening methods (e.g., self-sampling
as HPV L1 capsid protein [99–101] and E6 onco- or urine-based sampling) that can address chal-
protein detection [102,103] have been evaluated lenges in improving access to cervical cancer
in a limited number of small studies, but more prevention services in these settings.
evidence is needed to determine their utility.
Biomarkers in discovery & early
Biomarkers for low-resource settings validation phases
In the context of resource-constrained settings, Many discovery approaches for cervical cancer
the failure to establish and sustain cytology- screening biomarkers are currently underway.
based screening has necessitated research on Improved understanding of cancer epigenetics
operationally simple and less resource-intensive has led to a strong focus on methylation mark-
approaches for cancer prevention and control ers for biomarker development in many cancer
[104] . Visual methods such as visual inspection sites, including the cervix. In addition, several
with acetic acid and visual inspection with recurring chromosomal imbalances have been
Lugol’s Iodine provide immediate in vivo detec- observed for cervical precancer and are cur-
tion of visually apparent precancerous cervi- rently being explored as biomarkers. Finally,
cal lesions and the potential to link screening we briefly discuss potential markers earlier in
results and same-visit treatment by cryotherapy the development stage, such as miRNA and
(or appropriate referral for cryotherapy-ineligi- proteomic markers.
ble lesions). While visual inspection with ace-
tic acid/visual inspection with Lugol’s Iodine Epigenetic markers: DNA methylation
have been extensively evaluated [105,106] and Methylation of CpG sites within the genome
have high operational feasibility in the hands occurs at varying levels during carcinogenesis.
of nonphysician health providers, they miss While tumors are often hypomethylated in
anywhere between 20 and 50% of true dis- repetitive regions of DNA such as LINE ele-
ease due to variations in definitions of disease ments, promoter regions of tumor suppressor
positivity, inherent subjectivity in test results, genes may become hypermethylated, frequently
and challenges in quality assurance and control leading to decreased expression of important
[105,107] . There is a huge need for utilizing novel regulatory proteins (reviewed in [111]). Since
biologically-based approaches in resource- DNA methylation is a stable analyte that can
constrained settings of the developing world be detected in many biospecimens, and changes
for improving access and accuracy of screen- in methylation patterns that occur early in
ing [108] . careHPV™ is a new assay developed carcinogenesis are often retained in invasive
by Qiagen that is a low-cost adaptation of the tumors, they represent potentially clinically
Digene hc2 assay and can be performed rap- useful biomarkers.
idly (<2 h) without access to running water or Most work in the cervical cancer field has
electricity, an ideal solution for operation in focused on candidate genes that were identified
field settings [109] . This assay has been shown to by gene expression profiling in cervical cancer
have performance characteristics approaching cell lines and tissue or have been suggested to
those of hc2 [109] , and in conjunction with sim- play a role in tumor development in sites other
pler alternatives like visual inspection it may than the cervix. Very few studies have taken
permit effective single visit strategies (‘screen- advantage of microarray technologies or other
and-treat’) by same-day results and linkage to profiling approaches to identify differentially
cryotherapy [35,104] . Yet, further research on methylated genes [112–114] . Broadening the scope
adaptation of these strategies is needed to avoid of research to include previously unknown genes
overtreatment, given the different age distri- offers an opportunity to identify novel mark-
butions of HPV prevalence worldwide [110] . ers that could be useful clinically. In addition,
Novel biomarkers that reflect measurement of most methylation markers that have been stud-
an advanced disease process end point, such ied extensively come from studying the host
as overexpression of p16ink4a or HPV E6 pro- genome. However, there is growing evidence
tein detection [102,103] , are also being evaluated that methylation of HPV DNA may also be
in these settings, with the goal of achieving important in cervical carcinogenesis and could
an optimal balance of sensitivity and speci- provide additional biomarkers for screening
ficity for very infrequent testing. Additional and prognosis.

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 Review Sahasrabuddhe, Luhn & Wentzensen

Table 2. Methylation markers studied in cervical specimens.

Gene Number Methylation frequency Full name Biological function
of (number positive)
studies NL HGCIN† Ca
DAPK 22 0.068 0.296 0.582 Death-associated Serine-threonine kinase; positive mediator of
(33) (158) (659) protein kinase-1 IFN-g-induced apoptosis
RASSF1 17 0.031 0.102 0.141 Ras association Ras effector protein; microtubule regulation,
(10) (31) (175) (RalGDS/AF-6) domain cell migration, proliferation and apoptosis
family member-1
CDH1 15 0.159 0.129 0.521 Cadherin 1, E-cadherin Calcium-dependent cell
(37) (36) (456) adhesion glycoprotein
CDKN2A/p16 15 0.049 0.131 0.220 Cyclin-dependent Inhibits CDK4 kinase; regulation of cell-cycle
(17) (26) (187) kinase inhibitor 2A control in G1
MGMT 12 0.091 0.124 0.183 0-6 methylguanine- DNA repair
(33) (37) (124) DNA methyltransferase
RARB 12 0.045 0.130 0.343 Retinoic acid Regulates gene expression in response to
(15) (40) (169) receptor-b thyroid–steroid hormones
CADM1 10 0.256 0.385 0.657 Cell adhesion Intracellular adhesion
(43) (106) (236) molecule 1
FHIT 10 0.072 0.020 0.398 Fragile histidine triad Diadenosine 5´,5´´´-P1,P3-triphosphate
(21) (2) (268) gene hydrolase; purine metabolism
TIMP3 9 0 0.107 0.189 TIMP metallopeptidase Matrix metalloproteinase; degradation of the
(0) (6) (82) inhibitor 3 extracellular matrix
TERT 7 0.156 0.388 0.628 Telomerase reverse Enzymatic component of telomerase;
(12) (73) (120) transcriptase responsible for the addition of short repeats
to the ends of chromosomes or telomeres
CDH13 5 0.177 0.047 0.391 Cadherin 13, Calcium-dependent cell
(25) (7) (79) H-cadherin adhesion glycoprotein
PAX1 4 0 0.356 0.917 Paired box 1 Pattern formation during embryogenesis
(0) (36) (33)
TFPI2 4 0.200 0.342 0.721 Tissue factor pathway Regulation of plasmin-mediated
(20) (13) (88) inhibitor 2 matrix remodeling
CCNA 3 0.108 0.387 0.696 Cyclin A2 Activates CDK2 kinases; promotes G1/S and
(8) (24) (94) G2/M transitions
MAL 3 0.098 0.577 0.942 T-lymphocyte Candidate linker protein in T-cell signaling;
(4) (71) (227) maturation-associated implicated in myelin biogenesis and function
protein in the nervous system; formation,
stabilization and maintenance of
glycosphingolipid-enriched
membrane microdomains
TWIST 3 0.0928 0.403 0.362 Twist homolog 1 Transcription factor; differentiation and cell
(4) (27) (68) lineage determination
Inclusion criteria: genes that have been studied in normal, high-grade and cancer samples; genes that showed a low level of methylation (<20%) in normal
samples that increased in precancerous lesions and/or cancer samples; genes that have been reported in at least three studies; or genes that have been utilized in a
marker panel.
†
Includes CIN2, CIN3 and HSIL in calculations.
Ca: Cervical cancer; HGCIN: High-grade cervical intraepithelial neoplasia; NL: No lesion.

Host methylation regulators of cell growth and motility, there-

To date, methylation of many genes has been fore it is conceivable that they are more fre-
studied in cervical cancer. Table 2 �����������
lists indi- quently methylated, and presumably silenced,
vidual genes where the methylation status in in c­ervical cancer and its precursor lesions.
clinical samples has been published in at least A recent review of methylation markers in
three studies and summarizes the methylation cervical cancer detection reported that a total
frequency for each gene in normal, high-grade of 68 genes had been analyzed in 51 studies
and cancerous samples. There is a great diversity using clinical samples [115] . Since this review,
in the roles these genes play in normal cellular additional genes have been reported in the lit-
processes, ranging from apoptosis to cell–cell erature that show different methylation levels
interactions. In general, these genes are negative when comparing cervical cancers to normal

1090 Future Microbiol. (2011) 6(9) future science group

 Human papillomavirus & cervical cancer: biomarkers for improved prevention efforts Review

samples (Table 2) . However, identifying changes more frequently methylated in the later stages of
in methylation patterns in cervical precancers tumor progression and the methylation level has
will be important for biomarkers that can be been correlated to E6 mRNA expression [121,122] .
used in early detection. Some studies have In addition, methylation of CpGs within L1
shown that the frequency of DNA methylation have been shown to be elevated in high grade
of candidate genes increases with increasing lesions [123,124] . The functional relevance of this
severity of the cervical lesion, suggesting that p­henomenon is currently not known [125] .
these changes occur early in cancer develop- The data for methylation markers, both host
ment and are a potential source of biomarkers and viral, in cervical cancer screening has come
for early detection of cervical cancer (reviewed from small, heterogeneous studies, limiting the
in [115] and [116–120] , summarized in Table 2 ). evidence of their clinical utility. Although some
A few differentially methylated genes have small panels such as CADM1 and MAL have
been formally studied as diagnostic tools for promise as triage tests for HPV-positive women,
detection of cervical precancer (summarized the best panel or marker combination has yet
in Table 3 ), including single markers and marker to be identified and validated. The addition
panels. Table 3 summarizes the performance of of other genes such as DAPK, RARb, TWIST
panels of methylation markers for the detection or other viral markers to CADM1 and MAL
of cervical precancer. Although some candidates may be necessary to increase the diagnostic
have shown promising results, further studies are p­erformance of the panel.
needed to confirm that host methylation m­arkers
can be useful for cervical cancer prevention. Chromosomal abnormalities
Cervical carcinomas are characterized by a
Viral methylation high degree of genomic instability with many
Preliminary work has suggested that under- recurrent chromosomal amplifications and dele-
standing methylation of the HPV genome tions. Based on studies in clinical cervical can-
could lead to additional biomarkers for the cer samples, several regions are typically lost in
detection of cervical cancer and its progres- cervical carcinogenesis (2q, 3p, 4p, 5q, 6q, 11q,
sion. The promoter regions of E6 and E7 are 13q and 18q) while other regions are amplified

Table 3. Diagnostic performance of methylation markers.

Genes analyzed Study (year) Sample size End point AUC Sensitivity Specificity Ref.
(%) (%)
Individual markers
SOX1 Lai (2010) 185 CIN3+ 0.95 87.7 81.6 [142]

Lai (2008) 257 CIN2+ NR 26.0 97.0 [112]

LMX1A Lai (2010) 185 CIN3+ 0.90 76.7 88.4 [142]

Lai (2008) 257 CIN2+ NR 22.0 90.0 [112]

NKX6–1 Lai (2010) 185 CIN3+ 0.97 93.2 97.3 [142]
Lai (2008) 257 CIN2+ NR 58.0 67.0 [112]

WT1 Lai (2008) 257 CIN2+ NR 52.0 84.0 [112]

PAX1 Lai (2010) 185 CIN3+ 0.89 78.1 90.5 [142]

Huang (2010) 73 CIN3+ NR 71.4 93.3 [143]

Lai (2008) 257 CIN2+ NR 54.0 99.0 [112]

Marker panels
RARb/TWIST/MGMT Kim (2009) 209 HSIL 0.80 78.7 82.2 [144]
SOX1/HOXA11/CADM1 Apostolidou (2009) 59 HSIL 0.91 84.4 76.0 [145]

CCNA1/C13ORF18 Yang (2009) 185 CIN2+ NR 47.2 96.4 [146]

CADM1/MAL †
Overmeer (2011) 261 CIN3+ NR 70.0 78.0 [147]
Hesselink (2011) ‡ 250 CIN3+ 0.72 86.8 43.4 [148]
CDH13/DAPK/RARb/TWIST1 Feng (2005) 319 CIN3+ NR 74.0 95.0 [149]

Inclusion criteria: genes or panels were analyzed in clinical samples and diagnostic performance was reported.
†
Study populations were restricted to HPV-positive women only.
‡
Multiple cut-offs were reported in the article.
NR: Not reported.

future science group www.futuremedicine.com 1091

 Review Sahasrabuddhe, Luhn & Wentzensen

(1q, 3q, 5p and 8q; reviewed in [64]). Some of Proteomics

these alterations can be detected in p­recancerous The field of proteomics and the identification of
lesions (CIN3) [126–128] . differentially expressed proteins in biospecimens
Gain of 3q is the most consistently reported is a growing area of research. Most proteomic
chromosomal abnormality in cervical cancer. work in cervical cancer has focused on compar-
One gene within this region that is of particu- ing cancer specimens to normal samples to iden-
lar interest in carcinogenesis is TERC. A large tify potential markers for tumors. A serum-based
multicenter study in China recently confirmed study with 165 patients identified three peaks
previous small studies and showed that TERC by MALDI-TOF that were different between
amplification could serve as an effective triage cancer patients and healthy volunteers [139] . In
test for HPV-positive women who have ASC-US a validation data set, these biomarkers showed
or LSIL cytological diagnoses [129] . In addition, a sensitivity of 87.5% and specificity of 90% in
in a small study of women who had repeat pap the detection of cervical cancer.
smears available, TERC amplification was only Some studies have successfully used alterna-
seen in patients who progressed to a diagnosis of tive biospecimens for the identification of pro-
CIN3+ in follow-up tests from an initial diag- tein markers, including cervical–vaginal fluid
nosis of CIN1/2 [130] . A separate study showed from colposcopy exams and cervical mucus
that amplification of 3q had a high negative pre- [140,141] . suggesting that proteomic research does
dictive value for the development of CIN2+ in not need to be restricted to serum- or tissue-
women with LSIL cytology results [131] . Together based assays. However, most proteomic studies
this suggests that amplification of this region have been small, few have studied precancerous
could be useful in determining which women lesions, and any differentially expressed proteins
have clinically relevant HPV infections and need need to be validated in larger studies.
to be referred to coloposcopy and which women
can be monitored by continued screening. Future perspective
Other potentially important genes located Cervical cancer prevention is at a transition
within regions of gain or loss have not been from cytology-based screening programs to
definitively identified, however, Fitzpatrick HPV-based prevention. With primary preven-
et al. showed a correlation between expression tion using vaccines and secondary prevention
of mRNA and either loss (6p and 4q) or gain using a highly sensitive HPV DNA test with
(3q and 12q) of chromosomal DNA for genes long-term negative predictive value at hand,
within these regions in cervical cancer sam- extending screening intervals will be crucial
ples [132] . This could lead to the identification for these programs to work. New biomarkers
of other genes that are significant in cervical will be important to decide who among the
cancer development. HPV-positive women needs to be referred for
further evaluation or treatment. Large studies
On the horizon are currently underway for various triage bio-
miRNAs marker candidates. It can be expected that the
miRNAs, short noncoding RNAs, are respon- first screening programs based on primary HPV
sible for negatively regulating the expression of testing and new biomarkers as secondary tests
genes by binding to the mRNA and prevent- will be implemented in a few years. It will be
ing its translation. Abnormalities in miRNA important to reserve treatment for those women
expression patterns have been seen in a number who are at risk of developing cancer, rather than
of tumors and these changes have been sug- treating any high-grade lesion. Prospective bio-
gested to have prognostic value for other cancers markers may play an important role in these
(reviewed in [133]). Profiles of cervical tumors and therapy decisions. The implementation of new
cancer cell lines have identified miRNAs that prevention strategies will be very different in
have increased (miR-21, miR-127 and miR-199a) each healthcare setting; in a few years, we can
and decreased (miR-143, miR214, miR-218 and expect to see a wide variety of cervical cancer
miR-34a) expression in cancer compared with prevention programs existing in parallel.
normal tissue, suggesting a role for miRNAs
in cervical carcinogenesis [134–136] . Since these Conclusion
changes in expression are seen in early, precan- Large randomized trials have shown that pri-
cerous lesions [137,138] , they hold promise as bio- mary HPV screening for replacing cytology-
markers for cervical cancer screening; however, based screening is feasible and is likely to be
they have not been formally studied in this way. cost-effective because it allows the safe extension

1092 Future Microbiol. (2011) 6(9) future science group

 Human papillomavirus & cervical cancer: biomarkers for improved prevention efforts Review

of screening intervals after a negative HPV test. promising results obtained with a ruggedized
Disease-specific biomarkers such as p16ink4a , HPV test in low-resource settings are very excit-
HPV E6/E7 mRNA, or novel methylation ing. Similar to industrialized countries, low-cost
assays may serve as secondary markers after versions of disease-specific biomarkers could be
a positive HPV DNA test to identify women used to identify women who need immediate
with prevalent precancers who require immedi- treatment. In both high- and low-resource set-
ate colposcopy or treatment. At this point, these tings, incorporating biomarker data with a risk-
markers are not sufficiently validated for intro- based approach to screening will help to iden-
duction into HPV-based screening programs. tify assay c­ombinations that achieve optimal
The identification of prognostic biomarkers that risk stratification.
can predict progression to invasive cancers is an
important, but challenging area of biomarker Financial & competing interests disclosure
research. It is important to note that there is The authors have no relevant affiliations or financial
substantial heterogeneity in biomarker discovery involvement with any organization or entity with a
and validation studies. Even for some commer- financial interest in or financial conflict with the sub-
cial assays, the heterogeneity in study design and ject matter or materials discussed in the manuscript.
disease ascertainment limit the comparability of This includes employment, consultancies, honoraria,
results and the generation of high quality meta- stock ownership or options, expert testimony, grants or
analyses. Due to space constraints, we were patents received or pending, or royalties.
not able to cover the methodological aspects of No writing assistance was utilized in the production
cervical cancer biomarker validation [17] . The of this manuscript.

Executive summary
n Concerns about substantial cost burden associated with screening, limited accuracy of cytology, and complications of unnecessary
treatment have prompted research and development of more efficient approaches for cervical cancer prevention.
n New biomarkers may have potential use in primary screening, as triage tests for primary cytology screening and as triage tests for
primary human papillomavirus (HPV) screening.
n Detection of HPV E6/E7 mRNA and protein expression, as well as markers of cellular proliferation such as p16ink4a /Ki-67 immunostaining
have been widely validated and are in limited clinical use, mainly as triage markers after primary HPV DNA screening.
n Candidate biomarkers will be increasingly available for clinical validation through expansion in new technologies. Markers of viral and
host methylation changes, markers demonstrating chromosomal imbalances, miRNA, and proteomics markers are some of the most
likely candidates that may progress further in the developmental pipeline to clinical correlative studies.
n In both high- and low-resource settings, incorporating biomarker data with a risk based approach to screening will help to identify
assay combinations that achieve optimal risk stratification.

5. Jordan J, Arbyn M, Martin-Hirsch P et al. 9. Meijer CJ, Berkhof J, Castle PE et al.

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