Human papillomavirus testing for the detection of high-grade cervical intraepithelial neoplasia and cancer: final results of the POBASCAM randomised controlled trial (vol 13, pg 78, 2012)

Full text

www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
1
Articles
Human papillomavirus testing for the detection of
high-grade cervical intraepithelial neoplasia and cancer:
final results of the POBASCAM randomised controlled trial
Dorien C Rijkaart, Johannes Berkhof, Lawrence Rozendaal, Folkert J van Kemenade, Nicole W J Bulkmans, Daniëlle A M Heideman,
Gemma G Kenter, Jack Cuzick, Peter J F Snijders, Chris J L M Meijer
Summary
Background Human papillomavirus (HPV) testing is more sensitive for the detection of high-grade cervical lesions
than is cytology, but detection of HPV by DNA screening in two screening rounds 5 years apart has not been assessed.
The aim of this study was to assess whether HPV DNA testing in the first screen decreases detection of cervical
intraepithelial neoplasia (CIN) grade 3 or worse, CIN grade 2 or worse, and cervical cancer in the second screening.
Methods In this randomised trial, women aged 29–56 years participating in the cervical screening programme in the
Netherlands were randomly assigned to receive HPV DNA (GP5+/6+-PCR method) and cytology co-testing or cytology
testing alone, from January, 1999, to September, 2002. Randomisation (in a 1:1 ratio) was done with computer-
generated random numbers after the cervical specimen had been taken. At the second screening 5 years later, HPV
DNA and cytology co-testing was done in both groups; researchers were masked to the patient’s assignment. The
primary endpoint was the number of CIN grade 3 or worse detected. Analysis was done by intention to screen. The
trial is now finished and is registered, number ISRCTN20781131.
Findings 22 420 women were randomly assigned to the intervention group and 22 518 to the control group; 19 999 in the
intervention group and 20 106 in the control group were eligible for analysis at the first screen. At the second screen,
19 579 women in the intervention group and 19 731 in the control group were eligible, of whom 16 750 and 16 743,
respectively, attended the second screen. In the second round, CIN grade 3 or worse was less common in the intervention
group than in the control group (88 of 19 579 in the intervention group vs 122 of 19 731 in the control group; relative
risk 0·73, 95% CI 0·55–0·96; p=0·023). Cervical cancer was also less common in the intervention group than in the
control group (four of 19 579 in the intervention group vs 14 of 19 731; 0·29, 0·10–0·87; p=0·031). In the baseline round,
detection of CIN grade 3 or worse did not dier significantly between groups (171 of 19 999 vs 150 of 20 106; 1·15,
0·92–1·43; p=0·239) but was significantly more common in women with normal cytology (34 of 19 286 vs 12 of 19 373;
2·85, 1·47–5·49; p=0·001). Furthermore, significantly more cases of CIN grade 2 or worse were detected in the
intervention group than in the control group (267 of 19 999 vs 215 of 20 106; 1·25, 1·05–1·50; p=0·015). In the second
screen, fewer HPV16-positive CIN grade 3 or worse were detected in the intervention group than in the control group
(17 of 9481 vs 35 of 9354; 0·48, 0·27–0·85; p=0·012); detection of non-HPV16-positive CIN grade 3 or worse did not dier
between groups (25 of 9481 vs 25 of 9354; 0·99, 0·57–1·72; p=1·00). The cumulative detection of CIN grade 3 or worse
and CIN grade 2 or worse did not dier significantly between study arms, neither for the whole study group (CIN grade 3
or worse: 259 of 19 999 vs 272 of 20 106; 0·96, 0·81–1·14, p=0·631; CIN grade 2 or worse: 427 of 19 999 vs 399 of 20 106;
1·08, 0·94–1·24; p=0·292), nor for subgroups of women invited for the first time (CIN grade 3 or worse in women aged
29–33 years: 102 of 3139 vs 105 of 3128; 0·97, 0·74–1·27; CIN grade 2 or worse in women aged 29–33 years: 153 of 3139
vs 151 of 3128; 1·01, 0·81–1·26; CIN grade 3 or worse in women aged 34–56 years: 157 of 16 860 vs 167 of 16 978; 0·95,
0·76–1·18; CIN grade 2 or worse in women aged 34–56 years: 274 of 16 860 vs 248 of 16 978; 1·11, 0·94–1·32).
Interpretation Implementation of HPV DNA testing in cervical screening leads to earlier detection of clinically
relevant CIN grade 2 or worse, which when adequately treated, improves protection against CIN grade 3 or worse and
cervical cancer. Early detection of high-grade cervical legions caused by HPV16 was a major component of this benefit.
Our results lend support to the use of HPV DNA testing for all women aged 29 years and older.
Funding Zorg Onderzoek Nederland (Netherlands Organisation for Health Research and Development).
Introduction
Infection with high-risk types of human papillomavirus
(HPV) has a causal role in the development of cervical
cancer.1,2 This link has stimulated the development of
HPV DNA testing, which might be useful in primary
cervical screening.3,4 Furthermore, prophylactic HPV16
and HPV18 vaccines have been developed and
introduced in many countries as a primary prevention
method.5,6
Randomised controlled screening trials of HPV DNA
testing7–11 have shown a decreased detection of high-
grade cervical lesions at the second screening round
Published Online
December 15, 2011
DOI:10.1016/S1470-
2045(11)70296-0
See Online/Comment
DOI:10.1016/S1470-
2045(11)70334-5
Department of Pathology
(D C Rijkaart MD,
L Rozendaal MD,
F J van Kemenade MD,
N W J Bulkmans MD,
D A M Heideman PhD,
Prof P F J Snijders PhD,
Prof C J L M Meijer MD) and
Department of Epidemiology
and Biostatistics
(J Berkhof PhD), VU University
Medical Centre, Amsterdam,
Netherlands; Centre for
Gynaecological Oncology,
Amsterdam, Netherlands
(Prof G G Kenter MD); and
Cancer Research UK Centre for
Epidemiology, Mathematics
and Statistics, Wolfson
Institute of Preventive
Medicine, London, UK
(Prof J Cuzick PhD)
Correspondence to:
Prof Chris J L M Meijer, VU
University Medical Centre,
Department of Pathology,
PO Box 7057,
1007 MB Amsterdam,
Netherlands
cjlm.meijer@vumc.nl

Articles
2
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
compared with cytology alone. Although the screening
protocols, study end points, and interval between
screening rounds varied in these trials, the consistent
results suggest that HPV DNA testing oers better
protection against high-grade cervical lesions in second
screening rounds than do cytology-based screening
methods. Only one study10 was large enough to also
show protection against cervical cancer in the second
screening round.
We present the final results of the POpulation-BAsed
SCreening study AMsterdam (POBASCAM) trial, a
population-based, randomised controlled trial. Our
main goal was to assess whether HPV DNA testing
decreases detection of cervical intraepithelial neoplasia
(CIN) grade 3 or worse, of CIN grade 2 or worse, and of
cervical cancer, in the second screening round.
Additionally, to assess the most appropriate age at
which HPV DNA testing should start, we analysed
women invited for the first time (aged 29–33 years) and
older women (34–56 years) separately. We also assessed
how detection of high-grade lesions in two screening
rounds was associated with particular HPV genotypes.
Methods
Study design and participants
Patients were enrolled between January, 1999, and
September, 2002, as part of the nationwide cervical
screening programme. In the Netherlands, women are
invited for cervical cancer screening at 5 year intervals
starting in the year when they reach age 30 years and
ending in the year when they reach age 60 years. The
design, methods, and baseline results of the trial have
been described.7,12 Detection of neoplasia or cancer in
the first 48 months was classed as detected in the
first screening round and those detected during
48–108 months were classed as detected in the second
screening round. The cuto after 48 months was used
because women in the Netherlands are invited for a
new screen in the year they reach 30 years, 35 years,
40 years, and so on. Therefore, the actual screening
interval be tween invited smears is be tween 4 and
6 years. Women who had a history of CIN grade 2 or
worse, had abnormal cytology in the preceding 2 years,
or who had had a hysterectomy were excluded. Women
aged 57 years or older at baseline were also excluded
because they would not routinely receive a smear in the
second round. All participants provided written
informed consent. The trial was approved by both the
Medical Ethics Committee of VU University Medical
Centre and the Ministry of Public Health (The Hague,
Netherlands).
Randomisation and masking
LR randomly assigned women to the intervention or
control groups in a 1:1 ratio with computer-generated
random numbers after the cervical specimen had been
taken and administrative data entered into the central
study database. Neither the molecular technicians nor
the cytotechnicians had access to the central study
database, and so were unaware of a patient’s assignment.
Women in the intervention group were given their HPV
DNA and cytology result and managed accordingly. For
the control group, the patients and all medical personnel
were masked to the HPV DNA test results. At the
second screening round, women in both groups were
managed according to their newly obtained HPV DNA
and cytological test results. In both groups pathologists
were aware of the cytology result but not of the HPV
Normal or BMD Moderate dyskaryosis or worse
Cytology and high-risk
HPV testing at 6 months
Cytology and high-risk HPV
testing at 18 months
Colposcopy and biopsy
Normal cytology and
high-risk HPV positive
Normal or BMD, and high-risk
HPV negative
Moderate dyskaryosis or worse,
or high-risk HPV positive
Second screening round Colposcopy and biopsy
Normal or BMD high-risk
HPV negative
Moderate dyskaryosis or worse,
or BMD high-risk HPV positive
Cytology and high-risk
HPV testing at 6 months
Cytology and high-risk HPV
testing at 18 months
Colposcopy and biopsy
BMD
Normal or BMD, and high-risk
HPV negative
Moderate dyskaryosis or worse,
or high-risk HPV positive
Second screening round Colposcopy and biopsy
Second screening round
Normal cytology and
high-risk HPV negative
Colposcopy and biopsy
Moderate dyskaryosis or worse
Figure 1: Management of women in the intervention group at the first screening round
In the second screening round women in both groups were managed in the same way as the intervention group were in the first screening round. BMD=borderline or mild dyskaryosis.
HPV=human papillomavirus.

Articles
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
3
result. CIN (CIN grade 1 or worse) biopsies were
reviewed by two experienced cervical pathologists (FJvK
and LR) who were masked to the HPV and cytology
results.
Procedures
Women were screened at baseline with either combined
HPV DNA testing and cytology or cytology alone.12 At
the second screening round 5 years later, all women
were screened with both HPV DNA testing and cytology
so that we could detect high-grade CIN and cancer cases
in the control group that we would otherwise have
missed with cytology alone.
Conventional cytological smears were taken with a
Cervex-Brush (Rovers, Oss, Netherlands) or a cytobrush.
The brush was placed in a vial containing 5 mL
phosphate-buered saline for HPV DNA testing. Cy-
tology results were read according to the CISOE-A
classification,13 which can be roughly converted to the
2001 Bethesda system.14 Cytological results were
grouped as normal, borderline or mild dyskaryosis, or
moderate dyskaryosis or worse. In the 2001 Bethesda
system, borderline or mild dyskaryosis corresponds to
atypical squamous cells of unknown clinical
significance, atypical squamous cells and cannot rule
out high-grade squamous intraepithelial lesions, low-
grade squamous intraepithelial lesions, or atypical
glandular cells. Moderate dyskaryosis or worse
corresponds to both squamous and glandular high-
grade intraepithelial lesions.
HPV DNA testing was done by general primer
(GP5+/6+) PCR enzyme immunoassay,15 which detects
14 high-risk types (types 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 59, 66, and 68). Positive specimens were typed
by reverse line blotting.16
Figure 1 shows how patients were managed, in
accordance with Dutch guidelines,13,17 and has been
described in detail previously.7 Of the women who were
referred for colposcopy, colposcopy-directed biopsies of
suspicious parts of the cervix were taken for histological
examination, according to standard procedures in the
Netherlands.18 Colposcopies were not registered,
therefore, data of registered biopsy rates were used to
estimate the number of gynaecological tests.
Histology was examined locally and classified as
normal, CIN grade 1, 2, 3, or invasive cancer, according
to inter national criteria.19,20 Glandular intraepithelial
neoplasia grade 1, 2, and 3 were included in their
respective CIN categories. In the analyses presented
here, the original diagnoses were used. Cytology and
histology results were retrieved from the nationwide
registry of histopathology and cytopathology results
and, when necessary, from individual laboratory
records. The reviewed histological results for biopsies
of CIN grade 1 or worse are shown in the appendix.
The primary outcome measure was histologically
confirmed CIN grade 3 or worse, with a primary
endpoint of the number of CIN grade 3 or worse
detected. The secondary outcome measures were
cervical cancer and CIN grade 2 or worse. Cervical
cancer included squamous cell carcinoma,
adenocarcinoma, and adenosquamous cell carcinoma;
adenocarcinoma in situ were included in the CIN
grade 3 group.
Statistical analysis
The main analyses included all randomly assigned
women and were done by intention to screen. The
overall number of CIN grade 3 or worse identified in
each group was calculated for the first screening round,
the second screening round, and the two rounds
combined. For the second round, we included only
those women who did not have CIN grade 2 or worse at
baseline and who were eligible for subsequent
screening, because those with CIN 2 or worse were
managed according to current treatment protocols.
When enrolment was complete all women had the
opportunity of 108 months of follow-up. Events after
108 months were not included. Because HPV DNA
prevalence decreases with age and CIN grade 2 or worse
can spontaneously regress in young women, we
Figure 2: Trial profile
HPV=human papillomavirus. CIN2+=cervical intraepithelial neoplasia grade 2 or worse. *The major reason for women
not to be enrolled in our study was lack of time for general practitioners to explain the objectives of the study.
22
420 assigned to the intervention group
22
518 assigned to the control group
44
938 enrolled and randomly assigned
4282 not enrolled*
2421 excluded
7 hysterectomy
216 moderate dyskaryosis or worse
in preceding 2 years
201 missing HPV DNA test
1997 age ≥57 years at baseline
2412 excluded
9 hysterectomy
217 moderate dyskaryosis or worse
in preceding 2 years
186 missing HPV DNA test
2000 age ≥57 years at baseline
19
999 eligible 20
106 eligible
19
579 eligible 19
731 eligible
2829 did not attend
second screen
2988 did not attend
second screen
16
750 women attended second screen 16
743 women attended second screen
420 not eligible
267 CIN2+ in first screen
153 hysterectomy at first screen
375 not eligible
215 CIN2+ in first screen
160 hysterectomy at first screen
49
220 women eligible
First
screen
Second
screen
See Online for appendix
For the nationwide registry of
histopathology and
cytopathology results see
http://www.palga.nl

Articles
4
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
analysed women invited for the first time (aged
29–33 years) and older women (34–56 years) separately
to assess the most appropriate age at which HPV DNA
testing should start.
Reason for smear test was not recorded in the
nationwide registry of histopathology and cytopathology
results. The contribution of opportunistic screening
in women with a negative screening test at baseline
was estimated by the proportion of women who had an
additional smear before receiving a new invitation
after 5 years. Dierences in detection between the
groups were calculated with Fisher’s exact test. All
p values are two-sided. Analyses were done with SPSS
version 12.0.
A study size of 44 000 was calculated to be suciently
large to detect (with 80% power) a significant dierence in
the number of lesions CIN grade 3 or worse between
groups at the second screen after borderline or mild
dyskaryosis at baseline. The sample size was also sucient
to show a decrease at the second round of CIN grade 3 or
worse lesions in women in the intervention group who
had normal cytology at baseline compared with women in
the control group who had normal cytology at baseline.
For the power calculations, we assumed that 84·5% of the
Intervention group Control group
Total CIN0/1 CIN2 CIN3 Cervical
cancer
Total CIN0/1 CIN2 CIN3 Cervical
cancer
Baseline screen
Inadequate cytology
HPV DNA positive 2 0 1 0 0 0 0 0 0 0
HPV DNA negative 31 1 0 0 0 27 2 0 0 0
Normal cytology
HPV DNA positive 724 48 31 29 2 766 9 6 9 1
HPV DNA negative 18 562 218 7 3 0 18 607 233 4 2 0
Borderline or mild dyskaryosis
HPV DNA positive 185 29 24 34 3 192 30 25 29 1
HPV DNA negative 330 28 3 3 1 335 22 2 5 0
Moderate dyskaryosis or worse
HPV DNA positive 146 11 26 87 5 160 20 24 93 4
HPV DNA negative 19 6 4 3 1 19 4 4 6 0
Total 19 999 341 96 159 12 20 106 320 65 144 6
Subsequent screen
Inadequate cytology
HPV DNA positive 0 0 0 0 0 2 0 0 0 0
HPV DNA negative 21 0 0 0 0 27 0 0 0 0
No HPV DNA test 21 2 0 0 0 23 0 0 0 0
Normal
HPV DNA positive 284 18 10 9 1 272 26 14 14 0
HPV DNA negative 8941 98 6 1 0 8811 107 3 1 0
No HPV DNA test 7025 134 14 1 1 7108 162 7 2 1
Borderline or mild dyskaryosis
HPV DNA positive 67 20 9 12 0 77 16 12 15 1
HPV DNA negative 129 7 1 2 0 118 9 2 0 1
No HPV DNA test 164 23 14 7 0 200 22 9 11 1
Moderate dyskaryosis or worse
HPV DNA positive 31 3 5 20 0 41 0 6 27 3
HPV DNA negative 8 2 3 1 0 6 0 0 4 0
No HPV DNA test 59 10 10 26 2 58 8 9 29 4
No screening test* 2829 52 0 5 0 2988 59 0 5 3
Total 19 579 369 72 84 4 19 731 409 62 108 14
Both screens
Total 19 999 710 168 243 16 20 106 729 127 252 20
Cases detected by opportunistic screening are also included. CIN3=cervical intraepithelial neoplasia grade 3. CIN2=cervical intraepithelial neoplasia grade 2. CIN0/1=cervical
intraepithelial neoplasia grade 0 or 1. *Women who had no second round smear recorded.
Table 1: Cervical intraepithelial neoplasia and cervical cancers at baseline screen and subsequent screen

Articles
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
5
baseline smears were cytologically normal, that 14% of the
baseline smears were borderline or mild dyskaryosis, and
that 4% of the normal and 15% of the borderline or mild
dyskaryosis smears were positive for HPV DNA. The
prevalences of CIN grade 3 or worse in the second screen
were assumed to be 0·4% and 0·9% for women with
normal cytology and borderline or mild dyskaryosis at
baseline, respectively. Furthermore, the relative risk of
HPV DNA for CIN grade 3 or worse in the subsequent
round was assumed to be 13, which is the lower bound of
the 95% CI reported in a previous study,21 which provided
a con servative estimate for the required sample size. The
study was not powered to detect a dierence in CIN
grade 3 or worse at baseline. This trial is registered as an
International Standard Randomised Controlled Trial,
ISRCTN20781131.
Role of the funding source
The sponsors had no role in study design, data
collection, data analysis, data interpretation, or writing
of the report. The corresponding author had full access
to all the data in the study and had final responsibility
for the decision to submit for publication.
Results
Figure 2 shows the trial profile. The median age at
recruitment was 40·0 years (IQR 34·0–49·0 years) in
both groups. For women without CIN grade 2 or worse,
the median time since last cytological results was
5·0 years (4·4–5·5). Compliance to follow-up testing
(at least one repeat smear) was higher in the control
group (477 of 527 patients [91%]) than in the intervention
group (1007 of 1239 patients [81%]). The dierence in
com pliance was related to baseline cytology. In the
inter vention group, 456 of 515 (89%) women with
borderline or mild dyskaryosis at baseline, and 551 of
724 (76%) with normal cytology and a positive high risk
HPV test complied with follow-up.
Attendance for the second screen was not significantly
dierent between groups (16 750 of 19 579 [86%] patients
in the intervention group vs 16 743 of 19 731 [85%] in the
control group). Compliance to follow-up testing did not
dier signifi cantly between groups—539 (84%) of
644 patients in the intervention group and 564 (85%) of
667 in the control group complied. Attendance at the
second screen of cytologically negative, HPV DNA
positive women was much the same in the intervention
and control groups (553 of 724 [76%] women vs 588 of
766 [77%] women). 2424 of 18 562 (13%) women who
were negative at baseline in the intervention group and
2542 of 19 373 (13%) women who were negative at
baseline in the control group had an additional smear
before receiving a new invitation after 5 years
(opportunistic screening). Table 1 shows the number of
cervical lesions by study group, in relation to cytological
and HPV DNA results at the first and second screens.
Positive HPV DNA (masked in the control group) and
abnormal cytology at baseline were much the same for
the intervention and control groups.12 The number of
CIN grade 0 or 1 detected at the first and second screens
Intervention group (n [%; 95% CI]) Control group (n [%; 95% CI]) Intervention vs control
Risk difference (95% CI) Relative risk (95% CI) p value
Baseline screen*
Cervical cancer 12 (0·06%; 0·03–0·11) 6 (0·03%; 0·01–0·07) 0·03% (–0·01 to 0·07) 2·01 (0·76 to 5·36) 0·166
CIN3 or worse 171 (0·86%; 0·73–1·00) 150 (0·75%; 0·63–0·88) 0·11% (–0·07 to 0.28) 1·15 (0·92 to 1·43) 0·239
CIN3 159 (0·80%; (0·68–0·93) 144 (0·72%; 0·61–0·85) 0·08% (–0·09 to 0·25) 1·11 (0·89 to 1·39) 0·387
CIN2 or worse 267 (1·34%; 1·18–1·51) 215 (1·07%; 0·93–1·22) 0·27% (0·05 to 0·48) 1·25 (1·05 to 1·50) 0·015
CIN2 96 (0·48%; 0·39–0·59) 65 (0·32%; 0·25–0·41) 0·16% (0·03 to 0·28) 1·48 (1·09 to 2·04) 0·014
Subsequent screen†
Cervical cancer 4 (0·02%; 0·01–0·06) 14 (0·07%; 0·04–0·12) –0·05% (–0·09 to –0·01) 0·29 (0·10 to 0·87) 0·031
CIN3 or worse 88 (0·45%; 0·36–0·56) 122 (0·62%; 0·52–0·74) –0·17% (–0·31 to –0·03) 0·73 (0·55 to 0·96) 0·023
CIN3 84 (0·43%; 0·34–0·53) 108 (0·55%; 0·45–0·66) –0·12% (–0·26 to 0·02) 0·78 (0·59 to 1·04) 0·096
CIN2 or worse 160 (0·82%; 0·70–0·96) 184 (0·93%; 0·81–1·08) –0·12% (–0·30 to 0·07) 0·88 (0·71 to 1·08) 0·234
CIN2 72 (0·37%; 0·29–0·47) 62 (0·31%; 0·24–0·41) 0·05% (–0·06 to 0·17) 1·17 (0·83 to 1·65) 0·387
Both screens*
Cervical cancer 16 (0·08%; 0·05–0·13) 20 (0·10%; 0·06–0·16) –0·02% (–0·08 to 0·04) 0·80 (0·42 to 1·55) 0·617
CIN3 or worse 259 (1·30%; 1·15–1·46) 272 (1·35%; 1·20–1·52) –0·06% (–0·28 to 0·17) 0·96 (0·81 to 1·14) 0·631
CIN3 243 (1·22%; 1·07–1·38) 252 (1·25%; 1·11–1·42) –0·04% (–0·25 to 0·18) 0·97 (0·81 to 1·16) 0·752
CIN2 or worse 427 (2·14%; 1·94–2·35) 399 (1·98%; 1·80–2·19) 0·15% (–0·13 to 0·43) 1·08 (0·94 to 1·24) 0·292
CIN2 168 (0·84%; 0·72–0·98) 127 (0·63%; 0·53–0·75) 0·21% (0·04 to 0·38) 1·33 (1·06 to 1·68) 0·017
*n=19 999 in the intervention group, n=20 106 in the control group. †n=19 579 in the intervention group, n=19 731 in the control group. CIN3=cervical intraepithelial
neoplasia grade 3. CIN2=cervical intraepithelial neoplasia grade 2.
Table 2: Occurrences of high-grade disease

Articles
6
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
was similar in the intervention and control groups.
Most CIN grade 0 or 1 were detected after a negative
first screen whereas most CIN grade 2 or 3, and cancer
cases were detected after a positive first screen.
In the intervention group, 12 cancers (one adeno-
carcinoma and 11 squamous cell carcinomas) and five
adenocarcinomas in situ were identified in the first
screen; in the second screen, four cancers (one
adenocarcinoma and three squamous cell carcinomas)
and one adenocarcinoma in situ were identified. In the
control group, six cancers (two adenocarcinomas and
four squamous cell carcinomas) and five
adenocarcinomas in situ were identified at the first
screen; 14 cancers (two adenocarcinoma and 12
squamous cell carcinomas) and four adenocarcinomas
in situ were identified in the second screen.
Table 2 shows the number of high-grade cervical
lesions in each group at first and second screens. For
the first screen, detection of CIN grade 3 or worse did
not dier significantly in the intervention group
compared with the control group (p=0·239).
Additionally, the number of cervical cancer cases did
not dier significantly between the intervention and
control groups (p=0·166). However, significantly more
CIN grade 2 or worse were detected at baseline in the
intervention group than in the control group (p=0·015).
In the second screen, fewer CIN grade 3 or worse and
cervical cancers were recorded in the intervention
group than in the control group (p=0·023 and p=0·031,
respectively). However, there was no significant
dierence in detection of CIN grade 2 or worse in the
intervention group compared with the control group in
the second screen (p=0·234). When combining both
rounds, the detection rates in the intervention and
control groups were much the same for CIN grade 2 or
worse, CIN grade 3 or worse, and for cervical cancer
(table 2). However, over two screening rounds, a third
more CIN grade 2 were recorded in the intervention
group than in the control group (table 2).
In the first screen, two cancers in the intervention
group were missed by HPV DNA testing but had
abnormal cytology (in patients aged 32 years with FIGO
IB1 and aged 54 years with FIGO IA1). Three cervical
cancers were missed by cytology testing (one patient
aged 36 years and two patients aged 44 years, all three
with FIGO IB1), but tested positive for HPV DNA. Five
cervical cancers were diagnosed after borderline or mild
dyskaryosis cytology (in patients aged 30 years with
FIGO IB1, 32 years with FIGO IB1, 35 years with IB1,
40 years FIGO with 1B1, and 50 years with IA1), which
could have been missed if patients were not followed
up correctly. Only one woman who developed cervical
cancer (aged 59 years with FIGO IB1) had a negative
HPV DNA test at the second screen. She was also HPV
negative in the first screen.
For women positive for high-risk HPV, with normal
cytology at first screen, the number of high-grade
lesions diered between the intervention and control
group in both first and second screens (table 3). These
women also have a substantial risk of CIN grade 3 or
worse in the second screen in the intervention and
control groups. At first screen, CIN grade 3 or worse
was not significantly dierent between the intervention
and control groups for women with moderate
dyskaryosis or worse (96 of 165 women vs 103 of 179;
RR 1·01, 95% CI 0·84–1·21; p=0·9) and for women
with borderline or mild dyskaryosis (41 of 515 women vs
35 of 527; 1·20, 0·78–1·85; p=0·5). However, more CIN
grade 3 or worse lesions were detected in women with
normal cytology in the intervention group than in the
control group (34 of 19 286 patients vs 12 of 19 373; 2·85,
1·47–5·49; p=0·001). At the second screen, excluding
women who had CIN grade 2 or worse, or who had
hysterectomy at baseline, numbers of CIN grade 3 or
worse were not significantly dierent between study
groups in women with baseline moderate dyskaryosis
First screen Second screen
Total Cervical
cancer
CIN3 CIN2 Cervical
cancer
CIN3 CIN2
Intervention group
Inadequate cytology
HPV positive 2 0 0 1 0 0 0
HPV negative 31 0 0 0 0 0 1
Normal cytology
HPV positive 724 2 29 31 0 36 20
HPV negative 18 562 0 3 7 2 36 40
Borderline or mild dyskaryosis
HPV positive 185 3 34 24 1 9 8
HPV negative 330 1 3 3 0 1 2
Moderate dyskaryosis or worse
HPV positive 146 5 87 26 1 2 1
HPV negative 19 1 3 4 0 0 0
Total 19 999 12 159 96 4 84 72
Control group
Inadequate cytology
HPV positive 0 0 0 0 0 0 0
HPV negative 27 0 0 0 0 0 0
Normal cytology
HPV positive 766 1 9 6 8 57 18
HPV negative 18 607 0 2 4 4 32 36
Borderline or mild dyskaryosis
HPV positive 192 1 29 25 0 13 5
HPV negative 335 0 5 2 0 2 2
Moderate dyskaryosis or worse
HPV positive 160 4 93 24 2 3 1
HPV negative 19 0 6 4 0 1 0
Total 20 106 6 144 65 14 108 62
CIN3=cervical intraepithelial neoplasia grade 3. CIN2=cervical intraepithelial neoplasia grade 2.
Table 3: Cervical cancers and high grade intraepithelial neoplasia by test results in the first and second
screen rounds

Articles
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
7
or worse (three of 39 vs six of 48; 0·61, 0·16–2·30;
p=0·7) and in women with baseline borderline or mild
dyskaryosis (11 of 443 vs 15 of 461; 0·75, 0·35–1·62;
p=0·6). However, CIN grade 3 or worse diered between
groups in women with normal cytology at the first
screen (74 of 19 066 vs 101 of 19 196; 0·74, 0·55–0·99;
p=0·049).
The cumulative number of CIN grade 3 or worse
lesions detected over two screening rounds was much
the same for both study groups in women with moderate
dyskaryosis or worse at baseline (99 of 165 vs 109 of 179;
0·99, 0·83–1·17), women with borderline or mild
dyskaryosis at baseline (52 of 515 vs 50 of 527; 1·06,
0·74–1·54), and women with normal cytology at baseline
(108 of 19 286 vs 113 of 19 373; 0·96, 0·74–1·25).
Furthermore, the cumulative number of CIN lesions of
grade 3 or worse over two screening rounds was
significantly lower for women who tested negative for
HPV DNA at baseline in the intervention group than
for those who had normal cytology at baseline in the
control group (50 of 18 942 vs 113 of 19 373; 0·45,
0·32–0·63). Use of CIN grade 2 or worse as an outcome
measure gave much the same result (107 of 18 942 vs
177 of 19 373; 0·62, 0·49–0·79). Use of reviewed
histological results did not give dierent results
(appendix).
373 of 3139 (12%) women aged 29–33 years in the
intervention group and 379 of 3128 (12%) women aged
29–33 years in the control group had HPV DNA at
baseline (p=0·786). By contrast, HPV at baseline was
detected in 684 of 16 860 (4%) women in the intervention
group aged 34–56 years and in 739 of 16 978 (4%) of
women in the control group aged 34–56 years (p=0·176).
152 of 3139 (5%) women in the intervention group aged
29–33 years at baseline had biopsy samples taken,
compared with 129 of 3128 (4%) women in the control
group aged 29–33 years. In women aged 34–56 years,
baseline screen biopsy samples were taken from 456 of
16 860 (3%) patients in the intervention group and 407
of 16 978 (2%) in the control group. 712 of 3136 (23%)
women aged 29–33 years in the intervention group had
repeat screening test or histology, or both, after the
baseline round compared with 554 of 3128 (18%) in the
control group. In women aged 34–56 years, follow-up
was 3100 of 16 860 (18%) in the intervention group and
2887 of 16 978 (17%) in the control group. If women
with a negative first screen were not counted in the
numerator, follow-up among women aged 29–33 years
was 355 of 3139 (11%) in the intervention group and
172 of 3128 (5%) in the control group, for women aged
34–56 years follow-up was 843 of 16 860 (5%) in the
intervention group and 497 of 16 978 (3%) in the control
group. For the two screening rounds combined, the
two study groups did not dier significantly for CIN
grade 3 or worse and CIN grade 2 or worse for both
women aged 29–33 and those aged 34–56 (figure 3).
The ratio of CIN grade 3 or worse detection between
the intervention and control groups was 0·97 (102 of
3139 vs 105 of 3128; 95% CI 0·74–1·27) for women aged
29–33 years and 0·95 (157 of 16 860 vs 167 of 16 978;
0·76–1·18) for women aged 34–56 years. Ratios for CIN
grade 2 or worse were 1·01 (153 of 3139 vs 151 of 3128;
0·81–1·26) and 1·11 (274 of 16 860 vs 248 of 16 978;
0·94–1·32) for women aged 29–33 years and
34–56 years, respectively.
Finally, we compared the number of CIN grade 3 or
worse and CIN grade 2 or worse at first and second
screens for HPV16-positive and HPV16-negative women
(table 4). We only included women with a valid HPV
DNA test result. Individual HPV types other than
HPV16 were not analysed separately because of their
low prevalence in cervical lesions. For the first screen in
the intervention group 101 (59%) of 171 of the CIN
grade 3 worse and 135 (51%) of 267 of the CIN grade 2
or worse lesions were HPV16 positive. In women with
normal cytology, 23 (68%) of 34 of CIN lesions of
grade 3 or worse were HPV16 positive and in women
with abnormal cytology, 78 (57%) of 137 of CIN grade 3
or worse lesions were HPV16 positive. In the second
screen, fewer HPV16-positive CIN grade 3 or worse
0
50
100
0
20
40
60
80
100
120
140
160
180
150
200
250
300
I
n=3139
C
n=3128
I
n=3034
C
n=3032
I
n=3139
C
n=3128
I
n=16
860
Lesions (n) Lesions (n)
C
n=16
978
I
n=16
545
Second screen Both roundsFirst screen
C
n=16
699
I
n=16
860
C
n=16
978
CIN2
CIN3
Cervical cancer
A
B
Figure 3: Histological results for women aged 29–33 years (A) and
34–56 years (B), by study group and screening round
CIN2=cervical intraepithelial neoplasia grade 2. CIN3=cervical intraepithelial
neoplasia grade 3. I=intervention. C=control.

Articles
8
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
were detected in the intervention group than in the
control group (17 of 9481 vs 35 of 9354; RR 0·48, 95% CI
0·27–0·85; p=0·012) whereas detection of non-HPV16-
positive CIN grade 3 or worse did not dier (25 of
9481 vs 25 of 9354; 0·99, 0·57–1·72; p=1·00). The
proportion of HPV16 in HPV-positive CIN grade 3 or
worse and grade 2 or worse lesions was also lower in
the intervention group than in the control group but
the positivity ratio was not significantly dierent for
either CIN grade 3 or worse (17 of 42 vs 35 of 60, 0·69;
0·45–1·06; p=0·08) or for CIN grade 2 or worse (23 of
66 vs 42 of 92; 0·76, 0·51–1·14; p=0·17).
Discussion
Our results showed that fewer CIN lesions of grade 3 or
worse and cervical cancer were detected during second
screens in women who were screened for HPV DNA in
combination with cytology at first screen than those
who had cytology alone. Furthermore, the value of HPV
DNA testing is lent support by the finding that women
who tested negative for HPV DNA in the first screen
had significantly fewer CINs of grade 3 or worse
detected over two screening rounds than did women
with normal cytology at baseline. These findings accord
with previously reported data.4,7–11 However, our study
has longer follow-up than other studies did because the
screening interval in the Netherlands is longer than
that in other countries (5 years vs 3 years). This
dierence is important because it enables us to assess
whether a cervical lesion is persistent or regressive. Our
study also suggests that HPV screening does not have
to be postponed until age 36 years or older but can be
started at age 30 years because the cumulative two
screen detection of CIN of grade 3 or worse and grade 2
or worse did not dier between women aged 29–33 years
and women older than 33 years. Moreover, we showed
that early detection of high-grade cervical lesions caused
by HPV16 was a major component of the benefit of
adding high-risk HPV testing to cytology.
The interim analysis of 17 155 women from this trial
did not show a significant dierence between the
intervention and control groups in the number of
cervical cancers detected at the second screening
round.7 Our final analysis of the whole population was
large enough to identify a significant dierence in the
number of cervical cancers in the intervention group
versus the control group at the second screen. Such an
eect on cervical cancer in the second screening round
because of HPV DNA testing has only been shown in
one previous trial.10 The investigators reported no
cervical cancer in the second screen in 47 369 women
screened by HPV DNA testing, whereas nine cervical
cancers were recorded in 47 001 women in the control
group. Reduced mortality from cervical cancer because
of primary HPV DNA testing was also shown in a trial
in India.22 34 deaths from cervical cancer occurred in
the HPV DNA testing group (34 126 women) compared
with 64 in the control group (31 488 women).
The decreased relative risks of CIN grade 3 or worse
and cervical cancer in the second screening round
occurred after an increase in relative detection of CIN
grade 2 or worse in the baseline screen in the
intervention group compared with the control group.
By contrast with our interim findings,7 we failed to show
a significant dierence in the detection of CIN grade 3
or worse in the first screen in the whole study
population. However, our study was powered to detect a
significant dierence in the number of CIN grade 3 or
worse at the second round rather than at the first screen.
Also, in some other randomised trials no significant
dierence in detection of CIN grade 3 or worse at the
first screen occurred after HPV DNA testing, despite
decreased detection of CIN grade 3 or worse in the
Total number of women CIN3+ CIN2+
Intervention group
First screen
HPV16 333 (32%) 101 (63%) 135 (56%)
Inadequate 1 (0%) 0 1 (0%)
Normal 202 (19%) 23 (14%) 36 (15%)
BMD 55 (5%) 22 (14%) 30 (12%)
>BMD 75 (7%) 56 (35%) 68 (28%)
Other HPV type 724 (68%) 59 (37%) 107 (44%)
Inadequate 1 (0%) 0 (0%) 0 (0%)
Normal 522 (49%) 8 (5%) 26 (11%)
BMD 130 (12%) 15 (9%) 31 (13%)
>BMD 71 (7%) 36 (23%) 50 (21%)
Second screen
HPV16 100 (26%) 17 (40%) 23 (35%)
Normal 70 (18%) 6 (14%) 10 (15%)
BMD 19 (5%) 4 (10%) 5 (8%)
>BMD 11 (3%) 7 (17%) 8 (12%)
Other HPV type 282 (74%) 25 (60%) 43 (65%)
Normal 214 (56%) 4 (10%) 10 (15%)
BMD 48 (13%) 8 (19%) 16 (24%)
>BMD 20 (5%) 13 (31%) 17 (26%)
Control group
Second screen
HPV16 99 (25%) 35 (58%) 42 (46%)
Normal 53 (14%) 7 (12%) 10 (11%)
BMD 24 (6%) 8 (13%) 11 (12%)
>BMD 22 (6%) 20 (33%) 21 (23%)
Other HPV type 293 (75%) 25 (42%) 50 (54%)
Inadequate 2 (1%) 0 (0%) 0 (0%)
Normal 219 (56%) 7 (12%) 18 (20%)
BMD 53 (14%) 8 (13%) 17 (18%)
>BMD 19 (5%) 10 (17%) 15 (16%)
Data are n (%). CIN3+=cervical intraepithelial neoplasia grade 3 or worse. CIN2+=cervical intraepithelial neoplasia grade
2 or worse. BMD=borderline or mild dyskaryosis
Table 4: HPV types at baseline and subsequent screens and number of HPV-positive CIN grade 3 or worse
and CIN grade 2 or worse detected

Articles
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
9
second screening round.8,9 When we stratified our
analysis on the basis of baseline cytology, we did not
note a significant dierence in the occurrence of CIN
grade 3 or worse in women with borderline or mild
dyskaryosis or moderate dyskaryosis or worse but report
a significant dierence in the number of CIN grade 3 or
worse in women with normal cytology. The latter
finding matches our expectations because no additional
testing was recommended for women in the control
group with baseline normal cytology. The extra CIN
grade 3 or worse cases detected by HPV DNA testing
compared with cytology are the main benefit of using
HPV DNA testing for primary screening. The failure to
detect a relative dierence in the occurrence of CIN
grade 3 or worse could be related to the decision to
change the cytological classification criteria in 1996,
whereas the study was powered by cytological results
collected earlier. Notably, the use of more stringent
criteria for borderline or mild dyskaryosis and moderate
dyskaryosis or worse resulted in a decrease of borderline
or mild dyskaryosis in the Netherlands, from 11% in
1999 to 3% in 2004.13 This explains the low occurrence
of CIN grade 3 or worse in women with borderline or
mild dyskaryosis compared with other studies. The
cumulative number of women with CIN grade 3 or
worse and grade 2 or worse over both screening rounds
did not dier substantially between study groups, which
lends support to the idea that HPV DNA testing leads
to earlier detection of clinically relevant high-grade
lesions that, with the exception of a small number of
CIN grade 2 lesions, do not regress.
An important issue is the age at which HPV DNA
testing should be oered in primary screening.
HPV DNA prevalence is age dependent,23,24 therefore
HPV DNA testing in young women might have a
dierent risk–benefit ratio to HPV DNA testing in older
women. Ronco and colleagues10 reported that HPV DNA
testing in women aged 25–34 years could lead to
substantial overdiagnosis of regressive CIN grade 2 or
worse lesions, particularly when HPV DNA positive
women in this age group are directly referred for
colposcopy without further triage testing. Our results
suggest that with the triage algorithm used in this trial
HPV DNA testing in women aged 29–33 years does not
result in excessive diagnosis of lesions destined to
regress and lends support to the implementation of
HPV DNA testing in programmed cervical screening
starting at age 30 years.
The protective eect against CIN grade 3 or worse in
the second screen in the intervention group was largely
attributable to HPV16. HPV16 is the main genotype
present in cervical cancer, hence, the early detection of
HPV16-associated CIN grade 3 lesions is expected to
eventually have an eect on long-term outcomes such
as cancer morbidity and mortality. However, our study
was not large enough to provide detailed information
about the eect of other, non-HPV16 types, in the first
and second screening round. This investigation needs a
pooled analysis of many screening studies, which is
being done (pooling Swedscreen, NTCC, and data from
this study).8,10
Variation in histological classification of cervical
lesions between individual centres might be a limitation
of our study. However, we believe that the use of the
original diagnosis closely matches treatment guidance
and mimics future implementation of HPV DNA
testing in population-based screening programmes.
Moreover, use of histological classification obtained
after review by two experienced cervical pathologists,
although leading to more CIN grade 2 or worse and
CIN grade 3 or worse diagnoses, did not change our
conclusions.
A strength of our study is the large size of the trial
and the longitudinal design. We included all cytology
and histological follow-up data derived from the
nationwide registry within 9 years after the first screen.
Panel: Research in context
Systematic review
This trial was designed in 1997, when no data about performance of HPV testing in
population-based screening existed. The design was based on several findings. First, HPV
prevalence data obtained by general primer PCR in women with normal cervix, cervical
intraepithelial neoplasia (CIN) grade 1–3 and cervical cancer26 led to the hypothesis that HPV
testing by general primer PCR could be used for cervical cancer screening.27 After
improvement of our first generation general primer PCR by elongation of the primers and
addition of an easy readout,15 to allow high-throughput HPV detection, we applied this
GP5+/6+ general primer PCR in case-control studies, showing that high-risk HPV was present
in nearly all cervical carcinomas.28 Analysis of normal cervical scrapings of women who later
developed cervical cancer showed that the same HPV type as that in the carcinoma could be
found in the preceding normal smear.29 A subsequent prospective study on a gynaecological
outpatient population showed that the presence of a persistent HPV infection in women with
an abnormal smear was associated with progressive CIN disease.30,31 For the design of the
POBASCAM trial the Dutch health council, an independent advisory body of the government,
required that the study be done in the setting of the nationwide screening programme, which
has a screening interval of 5 years, which is why the results are only now available.
Interpretation
The final results accord with those of similar randomised controlled trials that used shorter
screening intervals, such as Swedscreen,8 ARTISTIC,9 and NTCC,10 as well as those of our
interim analysis,7 showing that HPV testing significantly reduces detection of CIN grade 3 or
worse in the second screening round relative to conventional cytology. Additionally, as was
the case in the NTCC trial, the final POBASCAM data also show that HPV screening protects
against cervical cancer better than does cytology alone. By contrast with other studies, CIN
was diagnosed by pathologists in daily routine practice rather than by a reviewed diagnosis,
to study the performance of HPV screening in a routine population-based setting. Review
of CIN diagnoses did not affect the results of the study. As such, the study can be classed as
an implementation study. Furthermore, the long screening interval as used in POBASCAM
allows assessment of whether a cervical lesion is persistent or regressive. Our study also
lends support to the notion that HPV screening does not have to be postponed until
patients reach age 35 years but can be implemented at age 30 years because the cumulative
detection of CIN grade 3 or worse and CIN grade 2 or worse across two screening rounds
does not differ between women aged 29–33 years and women 34 years or older.

Articles
10
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
Medical Centre (Amsterdam) for cytological testing and logistics, and
the administrative workers and the information technology team of
the Department of Pathology, VU University Medical Centre,
Amsterdam, for their supportive work. We also thank the Department
of Public Health and Social Medicine of Erasmus University
(Rotterdam) for help with the design of the trial, and the
gynaecologists in the study region for their contribution.
References
1 Walboomers JM, Jacobs MV, Manos MM, et al. Human
papillomavirus is a necessary cause of invasive cervical
cancer worldwide. J Pathol 1999; 189: 12–19.
2 Munoz N, Bosch FX, de Sanjose S, et al. Epidemiologic
classification of human papillomavirus types associated
with cervical cancer. N Engl J Med 2003; 348: 518–27.
3 Arbyn M, Sasieni P, Meijer CJ, Clavel C, Koliopoulos G, Dillner J.
Chapter 9: clinical applications of HPV testing: a summary of
meta-analyses. Vaccine 2006; 24 (suppl 3): S78–89.
4 Cuzick J, Clavel C, Petry KU, et al. Overview of the European and
North American studies on HPV testing in primary cervical
cancer screening. Int J Cancer 2006; 119: 1095–101.
5 Harper DM, Franco EL, Wheeler CM, et al. Sustained ecacy up
to 4·5 years of a bivalent L1 virus-like particle vaccine against
human papillomavirus types 16 and 18: follow-up from a
randomised control trial. Lancet 2006; 367: 1247–55.
6 Quadrivalent vaccine against human papillomavirus to prevent
high-grade cervical lesions. N Engl J Med 2007; 356: 1915–27.
7 Bulkmans NWJ, Berkhof J, Rozendaal L, et al. Human papillomavirus
DNA testing for the detection of cervical intraepithelial neoplasia
grade 3 and cancer: 5-year follow-up of a randomised controlled
implementation trial. Lancet 2007; 370: 1764–72.
8 Naucler P, Ryd W, Tornberg S, et al. Human papillomavirus and
Papanicolaou tests to screen for cervical cancer. N Engl J Med
2007; 357: 1589–97.
9 Kitchener HC, Almonte M, Thomson C, et al. HPV testing in
combination with liquid-based cytology in primary cervical
screening (ARTISTIC): a randomised controlled trial.
Lancet Oncol 2009; 10: 672–82.
10 Ronco G, Giorgi-Rossi P, Carozzi F, et al. Ecacy of human
papillomavirus testing for the detection of invasive cervical
cancers and cervical intraepithelial neoplasia: a randomised
controlled trial. Lancet Oncol 2010, 11: 249–57.
11 Anttila A, Kotaniemi-Talonen L, Leinonen M, et al. Rate of cervical
cancer, severe intraepithelial neoplasia, and adenocarcinoma in situ
in primary HPV DNA screening with cytology triage: randomised
study within organised screening programme. BMJ 2010; 340: c1804.
12 Bulkmans NW, Rozendaal L, Snijders PJ, et al. POBASCAM,
a population-based randomized controlled trial for
implementation of high-risk HPV testing in cervical screening:
design, methods and baseline data of 44,102 women. Int J Cancer
2004; 110: 94–101.
13 Bulk S, van Kemenade FJ, Rozendaal L, Meijer CJ. The Dutch
CISOE-A framework for cytology reporting increases ecacy of
screening upon standardisation since 1996. J Clin Pathol 2004;
57: 388–93.
14 Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda system:
terminology for reporting results of cervical cytology. JAMA 2002,
287: 2114–19.
15 Jacobs MV, Snijders PJ, van den Brule AJ, Helmerhorst TJ,
Meijer CJ, Walboomers JM. A general primer GP5+/GP6(+)-
mediated PCR-enzyme immunoassay method for rapid detection
of 14 high-risk and 6 low-risk human papillomavirus genotypes in
cervical scrapings. J Clin Microbiol 1997; 35: 791–95.
16 van den Brule AJ, Pol R, Fransen-Daalmeijer N, Schouls LM,
Meijer CJ, Snijders PJ. GP5+/6+ PCR followed by reverse line
blot analysis enables rapid and high-throughput identification of
human papillomavirus genotypes. J Clin Microbiol 2002;
40: 779–87.
17 van Ballegooijen M, Hermens R. Cervical cancer screening
in the Netherlands. Eur J Cancer 2000; 36: 2244–46.
18 Hopman EH, Rozendaal L, Voorhorst FJ, Walboomers JM,
Kenemans P, Helmerhorst TJ. High risk human papillomavirus
in women with normal cervical cytology prior to the development
of abnormal cytology and colposcopy. BJOG 2000; 107: 600–04.
The trial was done within the regular Dutch nationwide
screening programme. Together with the HC2 assay,
the GP5+/6+ PCR method is clinically validated and
performs much the same as HC2 in sensitivity,
specificity, and intra-laboratory and inter-laboratory
agreement.25 Therefore, our results can be considered
representative for organised cervical screening.
Our results are from a population-based screening
programme in which CIN diagnosis was made in
routine pathology laboratories. Therefore, our study
can be viewed as an implementation study,
representative for organised screening and as such
provides the strongest evidence to date in favour of
implementation of HPV testing in nationwide cervical
screening programmes (panel). On the basis of these
data the Health Council of the Netherlands, an
independent advisory body of the Dutch Government,
has issued advice to the minister of health to convert
the current cytology-based cervical screening
programme to an HPV testing-based programme
starting at 30 years. Additionally, oer of HPV testing
using self-collected cervico-vaginal samples to women
who do not respond to an invitation for cervical
screening results in a response of about 30%,32 showing
that a proportion of women prefer self-sampling to a
smear taken by a physician. Studies should investigate
the acceptance of this alternative for HPV testing in
regular attendees to the screening programme.
Ultimately, this option will likely increase attendance to
cervical screening programmes.
Contributors
CJLMM led the study and designed the study with LR. DCR, JB,
CJLMM, FJvK, PJFS, LR, and JC drafted the manuscript. DCR and JB
analysed the data. LR and NWJB managed the database. PJFS,
CJLMM, and DAMH did the HPV DNA testing. GGK liaised with the
gynaecologists. FJvK and LR oversaw cytological testing at the
individual laboratories. FJvK and LR reviewed histology. All authors
critically reviewed the manuscript and approved the final version.
Conflicts of interest
CJLMM is member of the scientific advisory board of Qiagen, has
received speaker’s fees from GlaxoSmithKline, and his institution has
received board membership and consultancy fees from Qiagen. PJFS
has received speaker’s fees from Roche and Gen-Probe. DAMH has
been an invited speaker for Roche. PJFS and CJLMM hold a patent for
HPV detection by GP5+/6+ PCR through Qiagen for which their
institution receives honoraria. CJLMM has also received honoraria and
holds a patent for promotor methylation of CADM1 as a specific
marker for CIN grade 2 or 3 or cervical cancer through MdxHealth, for
which his institution received honoraria. CJLMM, PJFS, and DAMH
are shareholders of Self-screen. JC is an ad-hoc adviser to Qiagen and
Roche. The other authors declare that they have no conflicts of interest.
Acknowledgments
The study was funded by Zorg Onderzoek Nederland (Netherlands
Organisation for Health Research and Development). We thank the
242 family doctors and their assistants, the Municipal Health Service
Southwest of Amsterdam, Medial, DHV Kennemerland-
Haarlemmermeer EO, and the nationwide registry of histopathology
and cytopathology. We also thank research sta and technicians of the
Unit of Molecular Pathology, VU University Medical Centre,
Amsterdam for HPV DNA testing, the cytotechnologists of the
Spaarne Ziekenhuis (Hoofddorp), Kennemer Gasthuis (Haarlem),
Leiden Cytology and Pathology Laboratory (Leiden), and VU University
For more on the Health Council
of the Netherlands see
http://www.gr.nl

Articles
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70296-0
11
19 Anderson MC. Premalignant and malignant squamous lesions of
the cervix. In: Fox H, Wells M, eds. Obstetrical and gynaecological
pathology, 4th edn. New York: Chruchill Livingstone, 1995: 292–97.
20 Wright TC. Precancerous lesions of the cervix. In: Kurman RJ, ed.
Blaustein’s pathology of the female genital tract, 4th edn. New
York: Springer Verslag, 1995: 248–57.
21 Rozendaal L, Walboomers JM, van der Linden JC, et al. PCR-
based high-risk HPV test in cervical cancer screening gives
objective risk assessment of women with cytomorphologically
normal cervical smears. Int J Cancer 1996; 68: 766–69.
22 Sankaranarayanan R, Nene BM, Shastri SS, et al. HPV screening
for cervical cancer in rural India. N Engl J Med 2009; 360: 1385–94.
23 Dillner J, Rebolj M, Birembaut P, et al. Long term predictive
values of cytology and human papillomavirus testing in cervical
cancer screening: joint European cohort study. BMJ 2008;
337: a1754.
24 Leinonen M, Nieminen P, Kotaniemi-Talonen L, et al.
Age-specific evaluation of primary human papillomavirus
screening vs conventional cytology in a randomized setting.
J Natl Cancer Inst 2009; 101: 1612–23.
25 Meijer CJ, Berkhof J, Castle PE, et al. Guidelines for human
papillomavirus DNA test requirements for primary cervical
cancer screening in women 30 years and older. Int J Cancer
2009; 124: 516–20.
26 Van Den Brule AJ, Walboomers JM, Du Maine M, Kenemans P,
Meijer CJ. Dierence in prevalence of human papillomavirus
genotypes in cytomorphologically normal cervical smears is
associated with a history of cervical intraepithelial neoplasia.
Int J Cancer 1991; 48: 404–08.
27 Meijer CJ, van den Brule AJ, Snijders PJ, Helmerhorst T,
Kenemans P, Walboomers JM. Detection of human
papillomavirus in cervical scrapes by the polymerase chain
reaction in relation to cytology: possible implications for cervical
cancer screening. IARC Sci Publ 1992; 119: 271–81.
28 Eluf-Neto J, Booth M, Muñoz N, Bosch FX, Meijer CJ,
Walboomers JM. Human papillomavirus and invasive
cervical cancer in Brazil. Br J Cancer 1994; 69: 114–19.
29 Walboomers JM, de Roda Husman AM, Snijders PJ, et al.
Human papillomavirus in false negative archival cervical
smears: implications for screening for cervical cancer.
J Clin Pathol 1995; 48: 728–32.
30 Remmink AJ, Walboomers JM, Helmerhorst TJ, et al.
The presence of persistent high-risk HPV genotypes in
dysplastic cervical lesions is associated with progressive disease:
natural history up to 36 months. Int J Cancer 1995; 61: 306–11.
31 Nobbenhuis MAE, Walboomers JMM, Helmerhorst TJM, et al.
Relation of human papillomavirus status to cervical lesions and
consequences for cervical-cancer screening: a prospective study.
Lancet 1999; 354: 20–25.
32 Gök M, Heideman DA, van Kemenade FJ, et al. HPV testing on
self collected cervicovaginal lavage specimens as screening
method for women who do not attend cervical screening: cohort
study. BMJ 2010; 340: c1040.

Comment
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70334-5
1
Published Online
December 15, 2011
DOI:10.1016/S1470-
2045(11)70334-5
See Online/Articles
DOI:10.1016/S1470-
2045(11)70296-0
How might HPV testing be integrated into cervical screening?
In The Lancet Oncology, final data from the POBASCAM
trial1 show the effectiveness of HPV testing for
cervical cancer screening. At baseline, HPV testing
identified 79 additional cervical precancers (cervical
intraepithelial neoplasia [CIN] grade 3) per 100 000
women and 30 additional cancers per 100 000 women
compared with screening using cytology only. During
the subsequent 5 years, the improved detection of CIN
grade 3 at baseline led to the detection of 24 fewer
precancers per 100 000 women per year and prevented
10 cancers per 100 000 women per year. Moreover,
with an assumption that all women in the intervention
group who tested negative for HPV and had a normal
Pap test returned in 5 years on average, we estimate a
very low cancer risk of 2·2 cancers per 100 000 women
per year, which shows that 5 year screening intervals
are safe.
POBASCAM reinforces findings from cohort
studies,2,3 clinical trials,4–6 and routine clinical practice7
by providing overwhelming evidence of the benefits
of inclusion of HPV testing in screening programmes.
However, clinical trials do not assess primary HPV
testing in isolation, but in the context of a full protocol
that determines management of all HPV-positive and
HPV-negative women. Detailed understanding of the
protocol is important to assess the feasibility of the
introduction of HPV testing in different settings and to
formulate guidelines for implementation.
The POBASCAM finding that is most immediately
translatable to screening programmes worldwide is the
5 year screening interval for women 30 years and older
who test HPV-negative with normal cytology. The low
estimated cancer risk for these women in POBASCAM
is much the same as the estimate from an analysis of
clinical practice in the USA—3·2 per 100 000 women
per year.7 These extremely low risks are consistent with
the fact that HPV infection is the cause of nearly all
cervical cancer and that cancer usually takes decades
to develop.8 We expect that almost every woman
who tests negative for HPV, irrespective of country or
screening protocol, has an extremely low risk of cancer
over 3 or 5 years.
How to manage HPV-positive women is less clear.
Most HPV infections will clear naturally and only a
minority of CIN grade 2 or 3 will progress to cancer.
Thus, an effective protocol that determines which
HPV-positive women should be referred to colposcopy
is crucial to prevent unnecessary colposcopies and
excisional procedures.9 The most aggressive protocol
refers all HPV-positive women to colposcopy, as was
done in the New Technologies for Cervical Cancer
screening (NTCC) trial5 of women 35 years and older.
POBASCAM used a more conservative protocol,
referring women to colposcopy only when they had
moderate dyskaryosis or worse cytology (equivalent
to high-grade squamous intraepithelial lesion)
at 6 months or moderate dyskaryosis or worse or
persistent HPV infection at 18 months. An aggressive
protocol has the advantage of potentially preventing
more cancer, whereas a conservative protocol has the
benefit of fewer diagnoses of intermediate endpoints
that might require unnecessary interventions.
In POBASCAM, ten cancers were prevented, with
only 32 additional CIN grade 2 or 3 diagnosed (table).
HPV-positive, Pap-negative women had eight of the
ten excess cancers, but also 26 of the 32 additional CIN
grade 2 or 3, confirming that HPV testing identifies
Pap-negative women at risk of cancer who are
challenging to manage. Conservative management
of HPV-positive women in POBASCAM resulted in
a smaller increase in CIN grade 2 detection, and a
greater increase in CIN grade 3 to grade 2 ratio than
in NTCC, probably because of regression of transient
CIN grade 2 lesions not destined to progress to cancer.
Incident
cancers in
Pap group
Incident
cancers in
HPV group
Cancers
prevented by
HPV group
Cumulative
CIN 2/3 in
Pap group
Cumulative
CIN 2/3 in
HPV group
Excess
CIN 2/3 in
HPV group
Inadequate
cytology
0 0 0 0 2 2
HPV negative,
Pap negative
4 2 2 74 86 12
HPV negative,
Pap positive
0 0 0 22 16 -6
HPV positive,
Pap negative
8 0 8 90 116 26
HPV positive,
Pap positive
2 2 0 193 191 –2
Total 14 4 10 379 411 32
A positive Pap result means any Pap abnormality. CIN 2/3=cervical intraepithelial neoplasia grade 2 or 3.
Table: Number of cancers prevented and excess diagnosis of CIN2/3 requiring excisional therapy for each
baseline HPV and Pap test result in POBASCAM

Comment
2
www.thelancet.com/oncology Published online December 15, 2011 DOI:10.1016/S1470-2045(11)70334-5
Despite the use of a more aggressive protocol, NTCC
prevented fewer cancers than did POBASCAM (nine in
total, but in more than twice the number of women
as POBASCAM), because NTCC had a lower baseline
cancer rate in the cytology arm than did POBASCAM
(19 per 100 000 vs 30 per 100 000). When a disease
is rare, fewer cases can be prevented, and prevention
of them might need a very aggressive protocol to
catch rare fast-progressing cancers, resulting in more
overtreatment.10
The POBASCAM trial shows that 5 year screening
intervals are safe, and that conservative management
of HPV-positive women can control excess CIN grade
2 or 3 while preventing cervical cancer. However,
how the POBASCAM protocol would perform in
other populations that have different baseline cancer
rates, compliance, and management infrastructure,
is unclear. Clinical management based on a woman’s
individual risk of cervical cancer might account for
population-specific factors that can have a substantial
effect on the performance of screening protocols.11
*Hormuzd A Katki, Nicolas Wentzensen
Division of Cancer Epidemiology and Genetics, National Cancer
Institute, Bethesda, MD, USA (HAK, NW)
katkih@mail.nih.gov
The authors declare that they have no conflicts of interest. HAK and NW are
supported by the Intramural Research Program of the National Cancer Institute,
National Institute of Health. The views expressed do not represent the views of
the US National Cancer Institute, the National Institutes of Health, the
Department of Health and Human Services, or the US Government.
1 Rijkaart DC, Berkhof J, Rozendaal L, et al. Human papillomavirus testing for
the detection of high-grade cervical intraepithelial neoplasia and cancer:
final results of the POBASCAM randomised controlled trial. Lancet Oncol
2011; published online Dec 15. DOI:10.1016/S1470-2045(11)70296-0.
2 Dillner J, Rebolj M, Birembaut P, et al. Long term predictive values of
cytology and human papillomavirus testing in cervical cancer screening:
joint European cohort study. BMJ 2008; 337: a1754.
3 Kjaer SK, Frederiksen K, Munk C, Iftner T. Long-term absolute risk of cervical
intraepithelial neoplasia grade 3 or worse following human papillomavirus
infection: role of persistence. J Natl Cancer Inst 2010; 102: 1478–88.
4 Mayrand MH, Duarte-Franco E, Rodrigues I, et al. Human papillomavirus
DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med
2007; 357: 1579–88.
5 Ronco G, Giorgi-Rossi P, Carozzi F, et al, on behalf of the New Technologies
for Cervical Cancer screening (NTCC) Working Group. Efficacy of human
papillomavirus testing for the detection of invasive cervical cancers and
cervical intraepithelial neoplasia: a randomised controlled trial. Lancet Oncol
2010; 11: 249–57.
6 Sankaranarayanan R, Nene BM, Shastri SS, et al. HPV screening for cervical
cancer in rural India. N Engl J Med 2009; 360: 1385–94.
7 Katki HA, Kinney WK, Fetterman B, et al. Cervical cancer risk for women
undergoing concurrent testing for human papillomavirus and cervical
cytology: a population-based study in routine clinical practice. Lancet Oncol
2011; 12: 663–72.
8 Schiffman M, Wentzensen N, Wacholder S, Kinney W, Gage JC, Castle PE.
Human papillomavirus testing in the prevention of cervical cancer.
J Natl Cancer Inst 2011; 103: 368–83.
9 Katki HA, Kinney WK, Fetterman B, et al. Premature conclusions on
HPV-only testing—Authors’ reply. Lancet Oncol 2011; 12: 993.
10 Castle PE, Katki HA. Benefits and risks of HPV testing in cervical cancer
screening. Lancet Oncol 2010; 11: 214–15.
11 Katki HA, Wacholder S, Solomon D, Castle PE, Schiffman M. Risk estimation
for the next generation of prevention programmes for cervical cancer.
Lancet Oncol 2009; 10: 1022–23.