Annals of Oncology Advance Access published January 19, 2014

review

Annals of Oncology 00: 19 , 2014

doi:10.1093/annonc/mdt538

Cervical cancer screening: on the way to a shift from

cytology to full molecular screening
M. G. Dijkstra1,2, P. J. F. Snijders1, M. Arbyn3, D. C. Rijkaart1, J. Berkhof4 & C. J. L. M. Meijer1*
1
Department of Pathology, VU University Medical Center, Amsterdam; 2Department of Obstetrics and Gynaecology, St. Lukas Andreas Ziekenhuis, Amsterdam, the
Netherlands; 3Unit of Cancer Epidemiology, Scientic Institute of Public Health, Brussels, Belgium; 4Department of Clinical Epidemiology and Biostatistics, VU University
Medical Center, Amsterdam, the Netherlands

Received 10 June 2013; revised 10 September 2013; accepted 29 October 2013

introduction
Currently, cervical cancer is the fourth leading cause of cancer
death in women worldwide, causing more than 275 000 deaths
annually. The disease has a very uneven global distribution; over
85% of cases are found in low-resource countries, with incidence
and death rates being the highest in sub-Saharan Africa, Central
America, South-Central Asia, and Melanesia [1, 2]. This imbalance in disease burden can be explained by differences in background risk [exposure to human papillomavirus (hrHPV)
infection], and the fact that cervical cancer is preventable by an
effective screening and intervention system. Therefore, the
lowest incidence and mortality rates are recorded in countries
where screening is available to women. The impact of population-based screening is reected in a substantial reduction in the
incidence of cervical cancer over the past 50 years in countries
with established cytology-based screening programs [36].
Especially, quality-assured population-based programs have
shown to be very effective [6]. However, cytology-based cervical
screening also has some limitations. The major problem is the
low sensitivity of a single smear to detect high-grade precursor
lesions (50%70%), which require frequent testing [7]. In addition, cytology has low reproducibility, leading to variable
*Correspondence to: Prof. Chris J. L. M. Meijer, Department of Pathology, VU University
Medical Center, PO Box 7057, 1007 MB Amsterdam, the Netherlands. Tel: +31-20-4444070; Fax: +31-20-444-2964; E-mail: cjlm.meijer@vumc.nl

accuracy [8, 9]. Moreover, by repeating cytology, the number of

false positives increases substantially over time [10]. Finally, the
decrease in the incidence of cervical cancer induced by
cytology-based screening is mainly restricted to squamous cell
carcinoma, whereas no change is observed in the incidence of
cervical adenocarcinoma [11, 12], suggesting that cytology fails
to detect adenocarcinomas and its precursors. Consequently,
there is a need for a better primary screening test, and thus a
new screening algorithm.

human papillomavirus testing in the

prevention of cervical cancer
cross-sectional sensitivity
Infection with hrHPV is a necessary event in the multistep
process of cervical carcinogenesis [13]. Thirteen hrHPV types
have been identied [14], of which HPV16 and HPV18 are the
most important types, causing 70% of squamous cell carcinomas, and >90% of adenocarcinomas [15, 16]. Recently, two
prophylactic vaccines against HPV16 and HPV18 (Cervarix
GSK and Gardasil Merck) have shown good protection against
vaccine type-related cervical intraepithelial neoplasia grade 2 or
worse (CIN2+) precursor lesions. However, cervical screening is
still required, because the current vaccines do not protect
against all carcinogenic HPV types. In addition, despite the high
vaccine uptake among women in countries with a school-based

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Cytology-based nation-wide cervical screening has led to a substantial reduction of the incidence of cervical cancer in
western countries. However, the sensitivity of cytology for the detection of high-grade precursor lesions or cervical cancer
is limited; therefore, repeated testing is necessary to achieve program effectiveness. Additionally, adenocarcinomas and
its precursors are often missed by cytology. Consequently, there is a need for a better screening test. The insight that infection with high-risk human papillomavirus (hrHPV) is the causal agent of cervical cancer and its precursors has led to
the development of molecular tests for the detection of hrHPV. Strong evidence now supports the use of hrHPV testing in
the prevention of cervical cancer. In this review, we will discuss the arguments in favor of, and concerns on aspects of implementation of hrHPV testing in primary cervical cancer screening, such as the age to start hrHPV-based screening,
ways to increase screening attendance, requirements for candidate hrHPV tests to be used, and triage algorithms for
screen-positive women.
Key words: hrHPV DNA, cervical cancer screening, triage, self-sampling

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Annals of Oncology

the rst round, has a lower specicity for CIN3+ than usually
expected [31]. Possible reasons for these discrepancies could,
rst, be attributed to overdiagnosis of lesions, as LBC was just
introduced at that time and secondly, to incomplete follow-up
of hrHPV-positive women with normal cytology, resulting in
lower detection rates of high-grade CIN in the hrHPV arm at
baseline [31, 32].

long-term protection
At present, four randomized trials conducted in Europe have
published longitudinal data on CIN3+ diagnosed at subsequent screening rounds, which took place in 35 years
(Figure 2) [2426, 28, 33]. All trials reported an 50% lower
CIN3+ detection rate in the second screening round among
women who were hrHPV negative at baseline than those who
had normal cytology. The pooled detection rate ratio of CIN3
+, of hrHPV testing versus cytology, was 0.42 [95% condence
interval (CI): 0.320.55]. The consistency of results was
reected by the absence of interstudy heterogeneity (P = 0.68).
Thus, a negative hrHPV test provides a better protection

Table 1. Trials comparing cytology and hrHPV testing in cervical cancer screening
Study

Description

Interval

Reference

POBASCAM

HPV (GP5+/6+-PCR) and cytology versus cytology

alone
HPV (HC2) combined with cytology (LBC) versus
cytology (LBC) alone
HPV (GP5+/6+-PCR) and cytology versus cytology
alone
HPV (HC2) alone versus HPV (HC2) and cytology
(LBC) versus cytology alone
HPV (HC2) and cytology versus cytology and HPV
(HC2) (randomized order of collection)
HPV (HC2) and cytology triage versus cytology
alone
HPV (HC2) versus cytology versus visual inspection
with acetic acid (VIA) versus no screening

5 years
3 years

Bulkmans et al. [24, 109]

Rijkaart et al. [33]
Kitchener et al. [25, 110]

35 years (by age)

Naucler et al. [26, 27]

3 years

Ronco et al. [28, 111, 112]

1 year

Mayrand et al. [29]

5 years

Leinonen et al. [113]

Shankaranarayanan et al. [41]

ARTISTIC
SwedeScreen
NTCC
CCCaST
Finnish screening trial
India screening trial

Study or Subgroup

Risk Ratio
M-H, Fixed, 95% Cl Year

Ronco 2006
Naucler 2007
Ronco 2008
Leinonen 2009
Kitchener 2009
Rijkaart 2012

1.24 [0.78, 2.00]

1.28 [0.90, 1.83]
2.06 [1.16, 3.68]
1.49 [0.88, 2.55]
0.94 [0.73, 1.21]
1.17 [0.93, 1.46]

Total (95% Cl)

1.18 [1.03, 1.35]

Risk Ratio
M-H, Fixed, 95% Cl

2006
2007
2008
2009
2009
2012

Total events
Heterogeneity: Chi2 = 7.69, df = 5 (P = 0.17); I 2 = 35%
0.1
1
0.01
Test for overall effect: Z = 2.36 (P = 0.02)
Favours experimental

10
100
Favours control

* Ronco et al. is restricted to women of 35 years or older

Figure 1. Detection rate ratio in screening with hrHPV versus cytology in the rst screening round of randomized trials for outcome CIN3+.

| Dijkstra et al.

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program [1719], the uptake remains suboptimal in, e.g. the

Netherlands, France, Germany, and the USA [2023].
The causal relationship between infection with hrHPV and
cervical cancer has stimulated the application of hrHPV DNA
testing, which has been proposed, either alone or in combination with cytology, as a means to improve existing cervical
screening programs. In the past 15 years, large randomized
trials designed to evaluate the performance of hrHPV testing
have provided important arguments for the implementation of
this assay as a primary screening tool (Table 1). First, ve of
these trials showed, in cross-sectional studies, that hrHPV
testing is 30% more sensitive in detecting CIN2+, and four of
these studies showed that it is also 20% more sensitive in
detecting CIN3+ (Figure 1). Most of these lesions are HPV16associated. The higher cross-sectional sensitivity was conrmed by two more studies analyzing the baseline data of
screening populations [29, 30]. The study published by
Kitchener et al. [25] showed somewhat different results; rst, it
demonstrated similar cross-sectional sensitivity for hrHPV
testing and liquid-based cytology (LBC) in primary cervical
screening, and secondly, the data suggest that LBC, as used in

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Annals of Oncology

Study or Subgroup
Naucler 2007
Kitchener 2009
Ronco 2010
Rijkaart 2012

Risk Ratio
M-H, Fixed, 95% CI Year
0.53 [0.29, 0.98]
0.52 [0.28, 0.97]
0.34 [0.15, 0.75]
0.39 [0.27, 0.56]

Risk Ratio
M-H, Fixed, 95% CI

2007
2009
2010
2012

Total (95% CI)

0.42 [0.32, 0.55]
Total events
Heterogeneity: Chi2 = 1.51, df = 3 (P = 0.68); I2 = 0%
0.01
0.1
1
Test for overall effect: Z = 6.37 (P < 0.00001)
Favours experimental

10

100

Favours control

Figure 2. Detection rate ratio of screening with hrHPV versus cytology in the second screening round, for outcome CIN3+, among women who were hrHPV
negative versus cytology negative in the rst screening round.

round by hrHPV testing represent non-regressing, clinically

relevant lesions [33].
Finally, an Indian cluster-randomized trial [42] found that, in
a low-resource setting, a single round of hrHPV screening was
associated with a signicant decline in the rate of advanced cervical cancers (FIGO stage II+), when compared with an unscreened control group [hazard ratio 0.47 (95% CI 0.320.69)].
No signicant reduction was observed in the study arms with
cytology or visual inspection with acetic acid screening.
Collectively, the available evidence indicates that sole hrHPV
testing should replace cytology as a primary screening tool in
cervical screening.

hrHPV detection methods

hrHPV detection methods include both HPV DNA assays and
E6/E7 mRNA assays. The drawback of hrHPV testing is that it
has an (apparently) unavoidable tradeoff between sensitivity and
specicity. Overall, hrHPV testing has a 3%4% lower specicity
than cytology [at cutoff atypical squamous cells of undetermined signicance or worse (ASC-US+)] [40] due to its inability to distinguish between persistent hrHPV infections
associated with ( precursor lesions of ) cervical cancer and transient hrHPV infections. The specicity could even be lowered
>25% when an ultra-sensitive hrHPV test was applied [43].
From a clinical point of view, testing for hrHPV is only useful
when a positive hrHPV test result is informative about the presence or absence of CIN2+ (clinical sensitivity and specicity).
Thus, in order to prevent excessive follow-up procedures for
women with transient hrHPV infections or hrHPV-positive
women without cervical lesions, candidate hrHPV tests to be
used for cervical screening should be clinically validated. Two
hrHPV DNA tests, i.e. HC2 and GP5+/6+, have shown, in large
clinical trials, to perform better in reducing the incidence of
CIN3+ and are thus considered as clinically validated prototype
assays. Guidelines for hrHPV test requirements and clinical validation were developed, based on the available data from large
prospective screening studies [44]. These guidelines can be used
to assess the clinical performance of a candidate HPV test, relative to one of two prototype hrHPV tests with proven good clinical performance (i.e. HC2 or GP5+6+-PCR) by a crosssectional clinical equivalence analysis in a screening setting [45].
In short, the candidate test should have a clinical sensitivity for

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against CIN3+, than a negative Pap smear. Several cohort

studies have also shown a consistently lower long-term cumulative incidence rate of CIN2+ among women negative for
hrHPV than those with normal cytology [30, 34, 35]. In addition, in two trials, the detection rate of cervical cancer at the
second screening round was signicantly lower among women,
who were hrHPV negative at baseline [odds ratio (OR): 0.19
(95% CI 0.070.53)] [28, 33]. Most importantly, in a pooled
analysis of four European trials following women for at least
two screening rounds, it was recently conrmed that hrHPVbased screening provides better protection against cervical
cancer than cytology [36]. Consequently, screening intervals
might be extended when primary hrHPV testing has been
introduced. Berkhof et al. [37] have shown that an extension to
6 or 8 years is possible without increasing the lifetime cancer
risk, using a simulation model. However, it makes sense to
extend the screening interval for hrHPV negatives only, as
hrHPV-positive women, even those with normal cytology,
have a non-negligible CIN3+ risk (5.2%) [33, 35]. The risk is
too high to delay follow-up to the next screening round (35
years) [38, 39], but too low to refer these women for immediate
colposcopy [40]. Therefore, hrHPV-positive women with normal cytology at baseline require further triage testing and/or
follow-up, which will be discussed below.
Another important and consistent nding in the ve trials
with combined hrHPV and cytology co-testing was that cotesting has virtually no additional value compared with single
hrHPV screening [40]. Although slightly more sensitive, no
signicant differences for CIN2+ or CIN3+ detection were
found [sensitivity ratio cytology and hrHPV versus hrHPV
alone: 1.06 (95% CI 0.961.18) for CIN2+, and 1.03 (95% CI
0.891.20) for CIN3+] [31]. A recent study from the USA also
showed that hrHPV and cytology co-testing have no advantage
over sole cytology screening [35]. Collectively, these data show
that sole hrHPV testing is sufcient for cervical screening, and
argue against the use of combined hrHPV and cytology cotesting in women aged 3060 years, as recently recommended
by the American Cancer Society (ASC) [41]. Moreover, the data
from the POBASCAM trial, in which hrHPV and cytology cotesting were used in both study arms in the second screening
round, show that the total number of CIN3+ detected over two
rounds is equal in both arms. These results indicate that at least
a part of the surplus of CIN2/3+ lesions detected in the rst

review

management of hrHPV-positive women

cytology and hrHPV genotyping
Epidemiological studies have estimated that HPV16 and HPV18
cause 70% of cervical cancers worldwide, and that the cumulative 10-year risk of CIN3+ in HPV16/18-positive women ranges
from 10% to 20% [60, 61]. These facts have led to several studies
evaluating the value of hrHPV genotyping, with or without cytology, to triage hrHPV-positive women.
In the VUSA-screen study, evaluating 14 triage strategies
[59], cytology triage at baseline and repeat cytology testing at 12
months emerged as the optimal strategy; it showed the highest
positive predictive value [PPV 37.5% (95% CI 32.642.6)] in
combination with a high negative predictive value (NPV) for

| Dijkstra et al.

CIN3+ [99.3% (95% CI 89.199.8)]. A CIN3+ risk of <2% (corresponding with a NPV for CIN3+ of >98%) was considered to
be acceptable for dismissal from further follow-up. This threshold was based on the 5-year CIN3+ risk of women with borderline or mild dyskaryosis (BMD) cytology at baseline, and
normal cytology at 6- and 18-month follow-up (1.2%), which is
presently accepted in the Netherlands [38].
In addition, we have recently carried out a post hoc analysis
of data from the POBASCAM trial [39] to evaluate useful triage
strategies, including HPV16/18 genotyping and cytology. Three
triage strategies met the criteria for both the NPV (98%) and
PPV (20%) that were set to minimize the risk of over investigation and excessively aggressive management; i.e. the NPVs
ranged between 98.1% and 99.6%, while the PPVs ranged
between 25.6% and 34.0%. The eligible triage strategies were:
(i) cytology and HPV16/18 genotyping at baseline without
repeat testing, (ii) cytology at baseline with repeat cytology
testing after 6 months, and (iii) cytology and HPV16/18 genotyping at baseline followed by repeat cytology examination at 6
months [39]. Furthermore, a nested evaluation of the Swedish
SWEDESCREEN study suggested to follow-up hrHPV-positive
women with normal cytology at baseline, by one repeat hrHPV
test [27]. This strategy showed comparable results in terms of
sensitivity for CIN3+ (96.0%), however, with somewhat lower
PPV (22.0%) than obtained with the preferred strategies in the
POBASCAM sub-study. Moreover, implementation of this strategy leads to a substantial increase in the colposcopy referral rate,
and thus possible overtreatment [39, 59].
Finally, Castle et al. [62] evaluated the performance of Cobas
HPV testing (Roche Molecular Systems, USA) as a primary
screening test among women aged 25 years and older. The preferred strategy in this sub-analysis of the ATHENA study was
cytology triage (threshold low-grade squamous intraepithelial
lesion or worse) in combination with the detection of HPV16,
HPV18, or both types, resulting in a sensitivity for CIN3+ of
72.2% (95% CI 66.477.4) and a PPV of 13.9% (95% CI 12.8
15.0). Although the authors recommended re-testing of screen
negatives after 1 year, because the sensitivity for CIN3+ was
<80% and the NPV <98%.
Thus, several triage strategies seem to be feasible, because the
results of the different studies also indicate that it does not
appear that the exact triage protocol has any effect on the
outcome. However, as preferences and the quality of cytology
will vary between countries, policymakers will have to weigh the
pros and cons of the different triage strategies when making a
choice. Especially, the balance between the safety of a triage
strategy (NPV) and the burden of screening on patients and
clinicians (PPV and referral rate) is important [63].

HPV mRNA, p16INK4a IHC, and methylation markers

In future cervical screening, it might be expected that the role of
cytology, as a triage test, will become more limited, as the
hrHPV test result may inuence the subjective reading of cytology. Promising biomarkers involved in cervical carcinogenesis have already emerged. Especially, biomarkers that indicate a
shift from the productive phase of hrHPV infection to the transforming phase are valuable. hrHPV E6/E7 oncogenes are highly
expressed in ( para)basal cells of high-grade CIN and interact

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CIN2+ not less than 90%, and a clinical specicity not less than
98% of that of the reference assays.
Thus far, three additional hrHPV DNA tests, i.e. Cobas 4800
Roche, Real-Time (RT) PCR Abbott Molecular, and
Papillocheck Bio-Greiner (when only 14 hrHPV types are considered), have fullled the criteria provided in these guidelines,
with sensitivities ranging between 100% and 95.8%, and specicities from 96.7% to 92.3% [4652]. Thus, these assays can be
considered as clinically validated for primary hrHPV-based cervical cancer screening.
In contrast to the HPV DNA tests, the APTIMA HPV Assay
(GenProbe) relies on aggregate detection of mRNA of 14
hrHPV types [53]. Two studies [54, 55] have evaluated the performance of this assay for primary screening compared with the
HC2 test, and the results were summarized in a recent review
[40]; the relative sensitivity ratio (outcome CIN2+) of APTIMA
versus HC2 was 1.02 (95% CI 0.861.20), and the relative specicity ratio was 1.07 (95% CI 1.051.08). Thus, the APTIMA
assay seems to perform with sensitivity closely to that of the
DNA test and with a possible slightly higher specicity. A recent
study comparing the APTIMA HPV Assay with GP5+/6+ PCR
showed similar results, and indicated that also the APTIMA
HPV Assay is clinically non-inferior to GP5+/6+ PCR with
respect to sensitivity and specicity for CIN2+ [56]. Another approach in trying to validate new hrHPV tests was used by
Cuzick et al. [57] in the predictors study: the sensitivity was primarily analyzed in a cytology-based referral population [58],
and specicity in a cytology-based screening population [57].
This study evaluated six HPV tests and established that four
tests (Roche Cobas, GenProbe APTIMA, Abott Real-Time PCR,
and (Becton, Dickinson and Company) HPV assay) achieved
the required sensitivity and specicity compared with HC2.
This would also qualify the BD test as clinically validated.
However, the predictors screening study only included cytologydriven CIN2+ lesion, which made the sensitivity criterion too
soft to consider it in line with the guideline described earlier
[44]. In addition, no details of any previous screening history of
participants were available. The use of validated HPV test assays
alone, however, is not sufcient to reduce the number of falsepositive tests among healthy women; triage testing of hrHPVpositive women is necessary to keep the number of invasive
follow-up examinations, and thus the costs within acceptable
limits [59].

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Annals of Oncology

expression. In many cancers, tumor suppressor genes were

found to be inactivated by hypermethylation of their promoter
region. Therefore, detection of hypermethylation of tumor suppressor genes involved in cervical cancer genesis may provide
powerful biomarkers for cancer detection [85, 86], especially as
methylation has been detected already at precancerous stages
[87, 88]. In fact, a recent study of Bierkens et al. showed that
methylation levels of two genes (i.e. CADM1 and MAL)
increased with the grade of underlying CIN and were highest in
carcinomas. Moreover, cervical scrapes of women with CIN 2/3
lesions with long-lasting hrHPV infections (5 years) had
higher methylation levels than those with a shorter duration of
preceding hrHPV infection (<5 years) [89]. These ndings indicate that at least lesions with a longer duration of existence are
detected by such methylation markers, further strengthening
their value for triage testing of hrHPV-positive cervical scrapes.
Recently, a study on scrapes from women participating in
screening was carried out to evaluate the value of an objective
real-time PCR assays that assess the methylation status of the
promoter regions of CADM1 and MAL to triage hrHPV-positive women for CIN3+ [90]. Because of the design of the study,
the results were compared with CIN3+ sensitivity and specicity
of cytology only, and of cytology combined with HPV16/18
genotyping. An optimal threshold resulted in a sensitivity of
84.2%, and a corresponding specicity of 52.5% for the methylation assay; for cytology, these were 65.8% and 78.8%, and for cytology with HPV16/18 genotyping, these were 84.2% and 54.0%,
respectively. Consequently, the authors concluded that, in
hrHPV positive women, this methylation marker panel was at
least equally discriminatory for high-grade CIN as cytology, or
as cytology with the detection of HPV16/18. These results indicate that complete cervical screening by objective, non-morphological molecular methods seems to be feasible. However,
further validation is needed before any of these tests can be considered for screening. Currently, a large randomized, controlled
trial to validate methylation markers to triage hrHPV-positive
women is being carried out in the Netherlands.

primary hrHPV testing: at what age

should we start?
Another important issue is the age at which hrHPV testing
should be offered for primary screening. Most experts agree
that, in women younger than 25 years of age, hrHPV testing is
not recommended, because the prevalence is very high, and at
this age, hrHPV infections are commonly transient.
Ronco et al. [28] reported that hrHPV testing in women aged
2534 years could lead to substantial overdiagnosis of regressive
CIN2+ lesions, particularly when hrHPV positives in this age
group are directly referred for colposcopy, without further triage
testing. The results from the POBASCAM trial [33] showed that
hrHPV screening does not have to be postponed until age 36
years or older, but can be started at age 30 years. The long-term
follow-up results of this study showed that the cumulative detection of CIN2+ and CIN3+ lesions over two screening rounds
did not differ between women aged 2933 years, and women of
34 years of age and older [33]. Thus, hrHPV screening does not
result in excessive diagnosis of lesions destined to regress, even

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with p53 and pRB, respectively; in this way, they interfere with
cell cycle control [64]. As a consequence, uncontrolled proliferation and chromosomal instability occur, resulting in additional
(epi) genetic changes [65, 66]. Therefore, detection of elevated
E6/E7 mRNA levels in cervical smears has been suggested to be
an attractive biomarker [67].
Two commercial hrHPV mRNA assays can be used for triage
testing, i.e. the PreTect HPV-Proofer (NorChip) and the
NucliSENS Easy Q HPV (bioMrieux). Both are based on the
same technology and are marketed under different brand names
in different countries. These assays detect HPV E6/E7 mRNA
from the ve most prevalent hrHPV types in cervical cancer
(HPV16, 18, 31, 33, and 45) [68]. A recent study [69] showed
that the HPV-Proofer assay is particularly of value to triage
hrHPV DNA-positive women with normal cytology, given their
markedly increased risk of CIN2+ in case of a positive mRNA
result [55% (95% CI 34%76%]. However, this study also
revealed that hrHPV-positive cytological normal women who
are mRNA test negative would still need follow-up, as their
CIN2+ risk was 20% (95% CI 7%33%). Thus, as a primary
screening test, the clinical applicability of these HPV mRNA
tests is insufcient
An alternative is the detection of cellular host genes that are
specically up-regulated and overexpressed, or silenced in cells
that have undergone the shift into the transforming phase of
hrHPV infection [70]. Cyclin-dependent kinase inhibitor
p16INK4a is up-regulated as a consequence of hrHPV E7 expression in proliferating cells [71, 72], and several studies have
shown that p16INK4a-based cytology, either alone or in combination with hrHPV testing, can detect underlying CIN2+ with
high sensitivity [7378]. However, the results for p16INK4a were
less favorable in the predictors studies [58, 79], comparing the
sensitivity and specicity of several tests for the detection of
high-grade CIN in a cytology-based referral population. In addition, even after p16INK4a immunostaining, still morphological
interpretation necessary to differentiate hrHPV-transformed
cells from endometrial cells with non-hrHPV induced p16INK4a
expression [80]. To overcome this limitation, a double staining
kit for p16INK4a and Ki-67 has been developed that allows simultaneous detection of p16INK4a and nuclear Ki-67 expression in
dividing cervical cells (CINtec Plus, MTM laboratories). The
potential of this double staining kit to identify women at risk for
underlying high-grade disease was shown in women with abnormal cytology [81, 82], as well as in hrHPV positives with normal
cytology, when used as a reex test [83]. At present, these good
results will need to be conrmed in prospective studies.
Furthermore, a recently published retrospective study evaluating
different triage strategies in a cytology-based referral population
suggested that further testing for p16INK4a and HPV16 genotyping may also be an important strategy to determine which
women are in need of referral. They advocate this strategy, especially for hrHPV screen positives with normal cytology [84].
Currently, these strategies should be evaluated in prospective
studies.
Although, a hrHPV infection can induce immortalization
and trigger chromosomal instability, additional changes in
oncogene expression and loss of function of tumor suppressor
genes are necessary, to obtain a full blown invasive cancer cell.
Methylation of CpG islands is an epigenetic modier of gene

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Annals of Oncology

in women in the 3034 years of age category. These ndings indicate that primary hrHPV screening should be recommended
for women aged 3060 years. Additional follow-up, after the age
of 60, is indicated for women with a positive hrHPV test at the
last screening round. The residual risk of CIN3+ is too high to
dismiss them from follow-up, even when triage testing in
hrHPV-positive women is negative [91]. These women can only
be discharged from further hrHPV testing after they have
cleared the virus. Future studies should investigate whether
methylation markers might be used as a more specic triage test
for hrHPV-positive women in the younger age range (below 30
years) to detect clinically relevant lesions, as high methylation
levels seem to be related to the degree and duration of underlying CIN and highest levels are found in carcinomas [89].

increasing the screening coverage

Table 2. Systematic reviews and meta-analyses of the literature

comparing self-collected and physician-taken samples for the
detection of hrHPV DNA
Reference

No. of studies
included

Kappa value for agreement

(range)

Ogilvie et al. [103]

Petignat et al. [95]
Schmeink et al. [114]

12 studies
18 studies
19 studies

0.451.00
0.500.82
0.450.81

| Dijkstra et al.

conclusion
All the evidence collected so far suggest that the time has come
for the implementation of hrHPV testing as a primary screening
test, as it provides a superior protection against cervical ( pre)cancerous lesions compared with cytology. hrHPV testing
detects 30% more CIN2+, and 20% more CIN3+ lesions in
women over 30 years of age. Although any hrHPV test used
should be clinically validated, hrHPV-based primary screening
should be implemented preferably within a population-based
screening program with a call and recall system. The somewhat
lower specicity, which may cause excess false-positive tests
among healthy women, can be overcome by triage of hrHPVpositive women. At this time, triaging these women by cytology
at baseline and repeat cytology testing after 6 months, possibly
in combination with baseline HPV16/18 genotyping, seems to
be very suitable for this purpose. Although, as preferences and
the quality of cytology will vary between countries, policymakers will have to weigh the pros and cons of the different
triage strategies when making a choice. In future screening,
however, it is likely that the role of cytology becomes more
limited and validated (molecular) biomarkers gain attention;
among these, p16INK4a/Ki-67 double staining and host genome
or viral DNA methylation markers appear to be promising.

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One other problem concerning the effectiveness of current cervical screening programs remains nonattendance [92, 93].
Especially nonparticipating women are at increased risk of
cancer [3, 94], as half of the cervical carcinomas are found in
non-attending women. Therefore, targeting non-attendees is
important in achieving optimal protection from screening programs.
Self-sampling is a less costly and less invasive collection
method [95], and several studies have shown that non-attendees
actually do take part in self-sampling studies [92, 9699]. Thus,
there is a basis for self-sampling in cervical cancer screening. In
addition, self-collection makes cervical screening accessible to
women in medium- and low-income countries [100102]. That
is why, in recent years, several studies have focused on the use of
self-collected samples for hrHPV testing.
In most studies, a moderate-to-good agreement between
hrHPV test positivity in self-collected and physician-taken
samples was found. The data have been summarized in (systematic) reviews and meta-analyses [95, 103], in which study parameters included concordance in hrHPV detection rates between
self- and physician-collected samples through kappa values,
which varied between 0.45 and 1.00 (Table 2). However, the
hrHPV test positivity rates varied across the studies. Most likely
due to differences in study populations (age range and country
of origin), the use of different collection devices for self- and
physician-sampling, as well as differences in hrHPV tests and
protocols. For example, in studies using the HC2 method, a
higher hrHPV detection rate in self- compared with physiciancollected samples was observed [97, 100, 104, 105]. The HC2
assay, however, is known to show some cross-reactivity to

low-risk HPV types [106], and these types tend to more commonly affect vaginal than cervical mucosa. In other studies, a
higher hrHPV detection rate was found in the physiciancollected cervical samples [107, 108]. Yet, overall the data show
that self-sampling is concordant with physician-sampling in
detecting hrHPV DNA.
However, before its use in cervical cancer screening, it is most
important to know how self-sampling performs with regard to
relevant disease outcomes. In other words, hrHPV self-sampling
procedures should be clinically validated in terms of sensitivity
and specicity to detect CIN2+ lesions. Various cross-sectional
studies have been carried out to compare the value of hrHPV
testing on self- with physician-collected samples to detect CIN2+,
and the results have recently been summarized by Snijders et al.
[99]. This review showed that hrHPV testing on self-sampled
specimen is as sensitive for CIN2+ in several studies as hrHPV
detection on physician-taken cervical samples, although sometimes less specic. However, the authors also reported that both
the type of self-sampling device and the type of hrHPV test
seem to inuence the clinical performance of hrHPV testing on
self-samples. Thus, for reaching clinical equivalence, in terms of
detecting high-grade CIN, between self- and physician-sampling, a certied combination of self-sampling device and validated hrHPV test is important. Overall, the data show that selfsampling for primary hrHPV testing offers possibilities to increase screening coverage by reaching non-responders, and in
future, might even be offered as a safe alternative screening
method to regular attendees.
Additionally, hrHPV-oriented cervical screening programs
will increase public awareness of the link between hrHPV and
cervical cancer, which may, in turn, lead to a higher uptake of
the prophylactic HPV vaccines and consequently a further reduction in cervical cancer incidence.

Annals of Oncology

As for the age to commence screening, we advocate the introduction of primary hrHPV testing in women from the age of 30,
to ensure that mostly clinically relevant, non-regressing highgrade lesions are detected. Furthermore, since a negative hrHPV
test offers a better protection (50%) against CIN3+ than normal
cytology, hrHPV testing can be implemented together with an
extension of the screening interval for hrHPV screen negative
women. In addition, to achieve optimal protection and screening coverage, self-sampling might be offered to non-attendees,
and in future possibly as an alternative method to regular attendees, under the condition that a validated combination of selfsampling device and hrHPV assay is used.
Collectively, these data show that the time has come to implement primary hrHPV testing in population-based cervical
screening, thereby offering women maximum protection against
high-grade CIN with less uncertainty and fewer screening
rounds.

PJFS declares that he has received speakers fee from Roche,

Abbott, GenProbe, and Qiagen. He is also shareholder of Selfscreen B.V. JB declares that he has received speakers fee from
Qiagen and a consultancy fee from GSK. CJLMM declares that
he has received speakers fee from Qiagen, Roche, and Merck.
He has received consultancy fees from Qiagen until 2010, and
he is minority shareholder of Self-screen B.V. All remaining
authors have declared no conicts of interest.

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