CHAPTER 3
METHODS AND TECHNIQUES OF CERVICAL SCREENING
Draft of 15. December 2003

CONTRIBUTORS: Marc ARBYN, Jean-Jacques BALDAUF, Daniel DA SILVA, Joakim
DILLNER, Joe JORDAN, Euphemia McGoogan, Pekka NIEMINEN, Julieta PATNICK,
Odette REAL, Guglielmo RONCO, Ulrich SCHENCK, Pär SPAREN and Elisabete
WEIDERPASS
1.
EXECUTIVE SUMMARY (MARC ARBYN) ........................................................................................ 73
2.
ASSESSMENT OF THE PERFORMANCE OF SCREENING TESTS: PRINCIPLES AND
CRITERIA (MARC ARBYN, GUGLIELMO RONCO)................................................................................. 75
3.
CONVENTIONAL CERVICAL CYTOLOGY (ULRICH SCHENCK, MARC ARBYN)................. 79
3.1. DESCRIPTION OF CONVENTIONAL CERVICAL CYTOLOGY ............................................................................. 79
3.1.1. Principles of conventional cytology .............................................................................................. 79
3.1.2. Reading a cervical smear .............................................................................................................. 79
3.1.3. Screening technique and localisation ........................................................................................... 79
3.1.4. Cell analysis: interpretation .......................................................................................................... 80
3.1.5. Adequacy of the sample ................................................................................................................. 81
3.1.6. Cytological findings ...................................................................................................................... 81
3.1.7. The cytological report ................................................................................................................... 82
3.1.8. Clinical applications of cervical cytology ..................................................................................... 82
3.2. PERFORMANCE OF CERVICAL CYTOLOGY ................................................................................................... 83
4.
LIQUID BASED CYTOLOGY (MARCARBYN, ULRICH SCHENCK, ODETTE REAL) ............. 85
4.1. DESCRIPTION .............................................................................................................................................. 85
4.2. RATIONAL FOR LIQUID BASED CYTOLOGY ................................................................................................... 85
4.3. RECENT REVIEWS, META-ANALYSES AND PILOT STUDIES ............................................................................ 86
4.3.1. Comparison of the accuracy of liquid-based cytology with the conventional Pap-smear ............ 86
4.3.2. Judgement of the adequacy of liquid-based cytology relative conventional Pap smears ............. 88
4.3.3. Demonstration pilot projects conducted in Scotland and England ............................................... 90
4.3.4. Influencing factors ........................................................................................................................ 92
4.3.5. Economical aspects of liquid based cytology ................................................................................ 92
4.3.6. Training, time trend effects ........................................................................................................... 92
4.4. PROPOSITIONS FOR FUTURE RESEARCH ....................................................................................................... 93
4.5. CONCLUSIONS ............................................................................................................................................ 93
5.
AUTOMATED CYTOLOGICAL SCREENING (PEKKA NIEMINEN, ULRICH SCHENCK,
MARC ARBYN).................................................................................................................................................. 94
5.1. DESCRIPTION OF AUTOMATED SCREENING DEVICES.................................................................................... 94
5.2. RATIONAL FOR AUTOMATED SCREENING .................................................................................................... 94
5.3. EVALUATION OF THE PERFORMANCE .......................................................................................................... 95
6.
COLPOSCOPY (JEAN-JACQUES BALDAUF, DANIEL DA SILVA, JOE JORDAN, MARC
ARBYN) ............................................................................................................................................................... 98
6.1. DESCRIPTION .............................................................................................................................................. 98
6.2. INDICATIONS FOR COLPOSCOPY .................................................................................................................. 99
7.
HPV DNA DETECTION (JOAKIM DILLNER, PÄR SPAREN, MARC ARBYN) ......................... 100
7.1. HUMAN PAPILLOMAVIRUS INFECTIONS ..................................................................................................... 100
7.1.1. Introduction ................................................................................................................................. 100
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7.1.2. HPV in relation to other risk factors for cervical cancer ............................................................ 101
7.1.3. Immunogenetic factors: an increased risk for cervical cancer? ................................................. 101
7.2. HPV TESTS: PRINCIPLES AND LABORATORY PRACTISES. .......................................................................... 103
7.2.1. Quality Control Criteria .............................................................................................................. 103
7.2.2. Types of HPV tests....................................................................................................................... 104
7.3. CLINICAL APPLICATIONS OF HPV TESTING................................................................................................ 104
7.3.1. Use of HPV testing in Primary Screening ................................................................................... 106
7.3.2. Use of HPV testing in HPV triaging of equivocal smears ........................................................... 115
7.3.3. Use of HPV testing in follow-up post treatment of CIN .............................................................. 116
7.4. IMPACT OF HPV VACCINATION ON DESIGN OF CERVICAL SCREENING PROGRAMS ..................................... 118
7.4.1. Immune response to HPV infection and immunization ............................................................... 118
7.4.2. Vaccine trials in humans ............................................................................................................. 119
7.4.3. Unresolved questions about HPV vaccine trials ......................................................................... 120
8.
GLOSSARIUM AND LIST OF ABBREVIATIONS............................................................................ 124
9.
REFERENCES ........................................................................................................................................ 124
9.1. EVALUATION OF TEST PERFORMANCE....................................................................................................... 124
9.2. CONVENTIONAL CERVICAL CYTOLOGY ..................................................................................................... 126
9.2.1. Historical background ................................................................................................................ 126
9.2.2. Screening technique .................................................................................................................... 126
9.2.3. Cytological diagnosis .................................................................................................................. 127
9.2.4. Quality assurance........................................................................................................................ 127
9.2.5. The cytological report ................................................................................................................. 128
9.2.6. Peformance of cervical cytology ................................................................................................. 128
9.3. LIQUID BASED CYTOLOGY ........................................................................................................................ 128
9.3.1. Systematic reviews concerning liquid based cytology ................................................................. 128
9.3.2. Other references on liquid based cytology .................................................................................. 129
9.4. AUTOMATED SCREENING .......................................................................................................................... 131
9.5. HPV DNA DETECTION (PARTIM) .............................................................................................................. 132
10.
APPENDICES ..................................................................................................................................... 139
10.1. APPENDIX 1: COLLECTION OF ADEQUATE PAP SMEARS (MARC ARBYN, JOE JORDAN, EUPHEMIA
MCGOOGAN, JULIETA PATNICK) .............................................................................................................. 139
10.2. APPENDIX 2: REPORTING SCHEMES FOR CERVICAL CYTOLOGY (ULRICH SCHENCK, MARC ARBYN) . 139
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1. Executive Summary (Marc ARBYN)
Screening for cervical cancer requires the use of a test, which is easy to perform by medical or
paramedical personnel, available at an acceptable cost, causing minimal discomfort to the
woman and has a high sensitivity and specificity for progressive intra-epithelial lesions (CIN).
Evidence of effectiveness should be based on its potential to reduce the morbidity and
mortality from cancer. High sensitivity for the detection of CIN is an insufficient criterion for
effectiveness, since CIN often regresses. High specificity is required to avoid anxiety,
unnecessary treatment and side effects.
The conventional Pap smear partially fulfils these criteria. Cytological screening every three
to five years can reduce morbidity and mortality from cervical cancer by 80% or more, if
offered in an organised setting. Nevertheless, the test-validity, in particular the test sensitivity
of the conventional Pap smear for CIN, is moderate: between 50 to 70% for CIN; but around
80% for high-grade CIN. Cytological screening in opportunistic settings is less effective and
in particular less cost-effective.
Occurrence of false-negative and unsatisfactory Pap smears was considered as a justification
to develop new technologies such as liquid based cytology and automated screening devices.
The quality of the evaluations of their performance was often poor, essentially limited to
cross-sectional cytological outcomes and rarely verified by a valid gold standard.
Liquid based cytology (LBC) was originally evaluated using the split-sample study design,
where first a conventional smear is prepared from the sampling device prior to the preparation
of an LBC smear. Later the direct-to-vial study design was applied which corresponds to the
intended use of LBC. The cost of an individual LBC test is considerably higher. Results
pooled from split-sample studies showed neither increased detection rates of high-grade
lesions, nor higher positive predictive value, sensitivity or specificity in the few studies with
systematic verification. However, in direct-to-vial studies significantly higher detection rates
of low- and high-grade cytological abnormalities were observed, whereas the positive
predictive value for histologically confirmed CIN2+ (grade II or more serious disease) was not
lower than for conventional cytology. These findings may suggest increased sensitivity
without significant loss in specificity. However, the level of evidence for this statement is
rather low because of insufficiently controlled and verified study designs. In general the
quality of LBC preparations has improved and their interpretation requires less time.
Randomised controlled trials comparing LBC versus conventional cytology, respecting the
rules of good diagnostic research, and using biopsy proven outcomes, are still needed before
superior performance of LBC can be considered as evidence-based.
New automated screening devices, targeting liquid based cytology, are being developed and
are expected to replace the machines that have been evaluated over the last decade. It was
considered inappropriate to pool information on devices that are no longer available.
One experience with the PAPNET device merits attention, since it was the only randomised
clinical trial comparing manual versus PAPNET interpretation of conventional Pap smears,
using cancer incidence as final outcome. The preliminary results of this trial did not show
differences in detection of cancer nor CIN2+. The specificity and positive predictive value
were similar as well.
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Ther is overwhelming evidence is available showing that infection with sexually transmittable
human papillomaviruses (HPV) is a necessary but insufficient aetiological condition for the
development of cervical cancer. Only high-risk HPV types are associated with cervical
cancer. Given this evidence, several applications for HPV DNA detection have been
proposed: (1) primary screening for oncogenic HPV types alone or in combination with
cytology; (2) triage of women with equivocal cytological results; (3) follow-up of women
treated for CIN to predict success or failure. Vaccination against high-risk HPV types is
another proposed preventive strategy.
A Europe Against Cancer-sponsored meta-analysis targeted the second application. From this
meta-analysis it was concluded that triage of women with equivocal cytological lesions with
HPV testing using the Hybrid-Capture II assay is more accurate than repeat cytology.
Another recent systematic review indicated that HPV DNA detection predicts treatment
failure more quickly than cytological follow-up.
The use of HPV detection in the context of primary screening is an object of debate. HPV
infections are very common and usually clear spontaneously. Therefore, detection of its DNA
includes a serious risk of over-diagnosis and psychological distress among HPV-positive
women. Nevertheless, the specificity of HPV detection can be enhanced by: screening in
women older than 30 years, where HPV clearance is less frequent; confirming persistence of
the same HPV type over a delay of 6-12 moths; identifying high viral loads and using the most
specific HPV detection methods. The high sensitivity of current HPV-DNA detection
methods yields very high negative predictive values even for adenocarcinoma precursors that
often escape cytological detection. Several capital questions remain unanswered, such as the
length of the increased negative predictive values in HPV-negative individuals; the definition
of the best strategy to follow-up HPV positives that are cytologically negative. Further
longitudinal research, by preference in an organised setting guaranteeing optimal follow-up,
and targeting public health relevant outcomes, before HPV screening can be recommended as
an alternative for, or in combination with, the Pap smear. Research should be completed with
mathematical modelling in order to define the best policy.
Progress has been made in the development of HPV vaccines. An overview is presented of
the current state of knowledge in this field.
Colposcopy is sometimes proposed as an alternative screening method, but its specificity is
too low to accept it for this use. Since colposcopy is an essential instrument to orient further
diagnostic exploration and treatment, it is covered more extensively in chapter 8.
In a first annex, a technical guideline is presented on the preparation of an adequate Pap
smear. In a following annex the cytological reporting forms that are currently in use in the
member states are added together with conversion tables allowing conversion to the current
Bethesda system. Chapter 3 ends with a discussion on a possible future European cytological
terminology.
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2. Assessment of the performance of screening tests: principles and criteria
(Marc ARBYN, Guglielmo RONCO)
The aim of cervical cancer screening is to identify progressive cervical intra-epithelial
neoplasia (CINa) and by their treatment prevent progression to invasive cancer [Morrison,
1992].
The effectiveness of a screening programme is determined by the programme sensitivity. This
programme sensitivity depends on the sensitivity of the chosen screening test, the natural
history of the disease, and the screening policy (the target age group, screening interval, and
procedures for follow-up of positive screens). The essential elements in the natural evolution
of the disease are the rates of onset, progression and regression of precursor lesions and the
distribution of their sojourn times. The mean sojourn time of CIN is at least 10 years and the
probability of detection increases as the preclinical phase progresses [Hakama, 1986; Van
Oortmarssen, 1991]. Therefore, repetition of a moderately sensitive screen test, such as the
Pap smear can reduce incidence of and mortality from cervical cancer to a low residual level
[Van Oortmarssen, 1992]. The reduction in the cumulative incidence of cancer is estimated to
be respectively 91 and 84% due to well organised cytological screening every 3 or 5 years
[Day, 1986; 1989; Van Oortmarssen, 1991].
In chapter 2, we learnt that the success of screening depends essentially on the
participation of the target population and the quality of the screening test and further on the
compliance and efficacy of treatment of screen-detected lesions. In this chapter we focus on
the performance of screening methods. We will describe and assess the performance of 5
main types of tests that are currently used in cervical cancer screening in Europe or that are
proposed as an alternative or supplement for current methods:
1. The conventional Pap smear
2. Liquid based cytology
3. Automated cytological screening
4. Colposcopy
5. Human papilloma virus DNA detection
A list of indicators for programme effectiveness, assessed by different study methods, is
enumerated in Table 1 and ranked from high to low according to the level of evidence that
such studies provide.
Table 1 Ranking of indicators by level of decreasing evidence for effectiveness of cervical cancer screening
methods according to the studied outcome and the used study design.
Outcome:
1.
2.
3.
4.
5.
6.
7.
Reduction of mortality from cervical cancer, life-years gained.
Reduction of morbidity due to cervical cancer: incidence of cancer (Ib+), Quality adjusted life-years
gained.
Reduction of incidence of cancer (including micro-invasive cancer).
Reduction of incidence of carcinoma in situ/severe dysplasia or cancer.
Reduction of incidence of high-grade CIN3+ or CIN2+.
Increased detection rate of CIN3+ or CIN2+.
Increased test positivity with increased, similar or hardly reduced positive predictive value.
In this chapter “CIN” (cervical intra-epthelial neoplasia) is used for histologically confirmed
lesions, while the SIL (Bethesda) terminology is used to describe cytological findings.
a
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Study designa:
1.
2.
3.
4.
5.
Randomised clinical trial, randomised population based trial.
Cohort studies.
Case-control studies.
Trend studies, ecological studies on routinely collected data.
Cross-sectional studies
5.a.1. Screening tests applied independently on the same study subjects;
5.a.2. Screening tests applied to separate but similar populations, historical comparison.
5.b.1.
5.b.2.
5.b.3.
5.b.4.
Complete golden standard verification of test negatives and positives; where by preference
verification is blinded to screen test results, allowing evaluation of test sensitivity and
specificity;
Complete verification of all test positives and a random fraction of test negatives;
Complete verification of all test positives and selective verification of screen negatives;
Incomplete selective verification of test positives and negatives.
5.c.1.
5.c.2.
Blinded gold standard verification without prior knowledge of screen test results;
Gold standard verification with prior knowledge of screen test results.
5.d.1.
5.d.2.
Randomly selected population or a continuous series of study subjects;
An arbitrarily chosen series of study subjects.
5.e.1.
5.e.2.
Population that is representative for the intended use of the test: (“spectrum of disease”) a
routine screening situation;
Setting with high risk women or setting referred women for previous abnormality or follow-up.
5.f.1.
5.f.2.
Reproducibility of the screen test result assessed;
Reproducibility of the screen test result not assessed.
Certain particular topics of Table 1 are highlighted below.
It must be stressed that the aim of screening is to prevent cervical cancer, not simply detect
pre-invasive lesions. A new screen test allowing (earlier) detection of more CIN does not
necessarily result in more pronounced reduction of cancer incidence since just additional nonprogressive lesions might be detected.
But, conducting randomised trials aiming to prove reduction in invasive cervical cancer
requires enormous financial resources and huge study populations to be followed for 10 or
more years including a high risk of contamination between the experimental and control arms.
Meanwhile the new technique might not be available anymore or obsolete. Therefore, certain
experts propose to study intermediate or surrogate outcomes (for instance outcomes 4 to 6 in
Table 1) and to simulate the most likely outcomes relevant to public health using
mathematical models.
The rate of progression of CIN and its detectability (or test sensitivity) by cytological
screening increases according to the severity of dysplasia [Canadian Task Force, 1976; Narod,
1991; Ostör, 1993; Melnikow, 1998; McCrory, 1999; Nanda, 2000]. Therefore detection of
a
Only controlled studies are considered, this means studies where two or more screening
methods are compared.
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CIN3+ constitutes a more pertinent outcome than CIN2+ [Sherman, 2002]. CIN1 is a much
less relevant outcome since most mild dysplasia regresses to normal [Ostör, 1993].
The assessment of the diagnostic validity, expressed in terms of sensitivity, requires the
explicit definition of test-thresholds for test positivity and disease. It can be evaluated by
application of screen tests to a relevant screening population followed by verification of all
subjects with an accurate golden standard. It can be assumed that histological examination of
material obtained by colposcopically directed biopsy, loop excision or endocervical curettage,
provides complete ascertainment of the true disease status. This might in fact not be true, but
independent verification with an imperfect golden standard will attract sensitivity and
specificity ratios (sensitivytest1/sentistivitytest2; specificitytest1/specificitytest2) towards unity.
Therefore observed accuracy differences are to be considered as minimum estimates.
Rarely colposcopy and histology can be applied to all screen negatives, which includes serious
risk of verification bias. Nevertheless, when 2 screen tests are applied to the same study
subjects and all subjects, positive for one or both tests, are verified with an acceptable gold
standard, unbiased estimation of the test positive predictive value, the relative sensitivity and
detection rate of true positives is possible [Schatzkin, 1987; Chock, 1997]. When a random
sample of screen-negatives (and or screen-test positives) are verified, an inferred sensitivity
and specificity can be computed [Begg, 1993; Choi, 1992; Irwig, 1994].
Given the low prevalence of disease, an approximated test specificity can be computed, even
without systematic verification of a random sample of test-negatives, from the ratio of the
number of test-negatives over the total number of study subjects minus the true positives
[Morrison, 1992]. (Specificityapprox= # test negatives / (N – # true positives); where N = the
number of all tested individuals).
The reliability or reproducibility of a test expresses the capacity to obtain the same test result
– correct or not – when the screening test is repeated on the same individual. The reliability
depends on the definition of distinct test criteria that can be applied by skilled personel. Poor
reproducibility automatically yields low sensitivity and specificity.
Once again, it must be repeated that the observation of increased sensitivity of a new test for
histologically confirmed CIN does not necessarily imply that its inclusion in a screening
programme will yield a reduction in incidence of lethal cervical cancer with respect to
conventional cytological screening. Nevertheless, when biological and epidemiological
arguments justify the assumption that the lesions detected in excess by the new method have a
substantial chance of progression (acceptable longitudinal positive predictive value) and that
screen negatives have a substantially lower chance to develop cancer in the future (higher
longitudinal negative predictive value), planning of the new test in a randomised populationbased trial in an organised setting can be considered (see chapter 2). Simulation models
should be performed to define the best policy for the new test.
Until now we studied essentially programme effectiveness stressing test sensitivity. Cervical
cancer screening addresses large populations and are therefore extremely costly. Costs are
largely determined by the test specificity. An overview of the cost components attributed to
screening is presented in Table 2.
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Table 2. Overview of cost components of a screening programme
1.
2.
3.
4.
5.
6.
Cost price of the screen-test (investment and recurrent costs); fees of health professionals (time for
preparation, interpretation of the screen test, documentation, training); logistical costs (transport,
processing, storage); administrative costs (invitation, registration and analysis of data).
Specificity of the screen test: cost of follow-up and treatment of women with false-positive results or
having non-progressive screen-detected lesions (over-diagnosis).
Sensitivity of the screen test (longitudinal): cost for follow-up and treatment of true positives; this cost
must be off-set by cost savings in avoided treatment of advanced disease.
Human costs: time spent by women to be screened, anxiety and discomfort for follow-up and/or
treatment of women with true and false-positive results and consequences of delay in detection of cancer
in false-negative women.
Specificity of quality control, triage and diagnostic follow-up procedures, contributing to increased
positive predictive value and savings by avoiding treatment of false-positive women.
Quality of screen test procedures; satisfactory rate influencing the need for repeat tests.
A small decrease in specificity can have dramatic consequences on costs. The number of
additional false positives is computed from nearly the complete target population, since the
prevalence of progressive cervical cancer precursors is low. Nevertheless, increasing the
screening interval can compensate the consequences of decreased specificity. Reliable
mathematical models can be used to estimate the final outcome per unit of cost.
In sub-chapter 3, we will discuss the current standard screening test, the conventional
Papanicolaou test.
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3. Conventional cervical cytology (Ulrich SCHENCK, Marc ARBYN)
3.1. Description of conventional cervical cytology
3.1.1. Principles of conventional cytology
Cytology can be defined as the science of cells. In the clinical context this is generally
understood as cytology applied for diagnostic purposes. Several synonyms are used: cytology,
diagnostic cytology, clinical cytology and cytopathology. In this chapter the term cervical
cytology is preferred to other terms like vaginal cytology, gynaecologic cytology, Pap test, and
Pap smear.
Cells are sampled with a sampling device from the surface of the uterine cervix and the
cervical canal. Cells are either directly smeared on a glass slide or deposited on a slide after
being first transferred to a liquid medium (see technical guideline in appendix 1: collection of
adequate Pap smears). For visual evaluation by the human observer the cells must be stained.
The cells are then analysed using a microscope.
3.1.2. Reading a cervical smear
Reading a cervical smear is a rather complex procedure. Primarily it can be understood as a
search procedure including single cell classification and a slide interpretation. It is clear that
hundred thousands of cells cannot all be evaluated visually in detail. The number of atypical
cells in a smear may be quite low, especially in low-grade intraepithelial lesions. The problem
of searching for a few atypical cells in a large area has been the basis for the development of
the cytotechnologists' profession, and a thorough visual screening of cervical smears is still
the basis for all cervical cancer screening programs. This chapter will address both the
localisation phase and the interpretation phase of cervical cytology. In reality both cannot be
easily separated.
3.1.3. Screening technique and localisation
Magnification: The resolution of the unaided human eye is about 0.1mm, 100µ. Considering
a nuclear size of 10µ, a ten power magnification is about the minimal enlargement required if
regular sized nuclei are to be detected at all. At this magnification no nuclear detail can be
recognised. For screening purposes generally another 10 power magnification is used. With
this set up we end up with a standard of a 10 power objective and a 10 power eye piece
magnification resulting in a 100 power total magnification, which is today the standard for
screening purposes. With this magnification nuclear features are mainly size and contrast,
wheras structural resolution is very poor even after foveal fixation. Lower magnification can
be used for orientation but not for screening in gynaecological cytology. Higher magnification,
25x and 40x are used to view objects of interest in more detail.
Slide movement: Generally, the screening of a case starts on one of the edges of the cover
glass. After the inspection of the field of view, the observer passes on to the next field of view
with a quick movement of the stage. This process of alternating movements and stops is
continued in the same direction until the opposite side of the cover slip is reached. Here the
observer moves to the next line where the screening is continued in the opposite direction. In
this way the slide is screened in a "meander"-like fashion until the total area of the slide has
been screened.
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Physiology of visual microscopic perception: The screening process can be understood as
follows. During about 180 msec the slide is moved from one field of view to the next. During
this time there is no foveal fixation. The new field of view is examined during the latency
period by peripheral vision. If no conspicuous object is found, after about 230 msec the
microscope stage is moved to the next field of view. If there is a conspicuous object, it will be
fixed by the fovea after a very rapid eye movement, a saccade. If necessary, in the same field
of view several objects will be fixed, each after a saccadic eye movement. Then the stage will
be moved to the next field of view. This process shows some obvious limitations in screening
performance. Only a limited part of the specimen area is analysed with stationary fields of
view. During stage movement no fixation takes place. Most of the area can be covered only by
peripheral vision. In the periphery of the fields of view foveal fixation is found less frequently.
Screening duration: The relation of total screening time, number of fields of view and the
slide area in stationary fields of view can be calculated. It is evident that there is a correlation
between total screening time and the specimen area that can be overseen during the stops in
the screening process. There is no doubt that with less screening time per specimen, only part
of the total area is seen. The use of large-field binoculars (e.g. 40° visual angles) does not
really solve the problem since the detection threshold of peripheral vision increases towards
the margin of the fields of view. The use of miniature cover glasses also does not seem
acceptable. The use of special cell preparation techniques like liquid based cytology resulting
in deposition of the representative sample with a randomised distribution in a limited slide
area has been discussed and is at present the only acceptable approach to reduce the deposition
area substantially. If the time spent by a cytotechnologist on a case is evaluated, not only the
screening time but also some time for documentation of the screening results has to be taken
into account. If a cytotechnologist needs on the average five minutes per slide and one minute
for documentation, he or she will be able to read 10 cases per hour and 60 cases per day in six
hours spent at the microscope. Of this time, about 60 minutes will be used for reading patient
documents and filling in forms. Organisation of the lab should focus on keeping the inter-case
interval short in relation to the time at the microscope. Screening during a shorter period will
yield substantially lower sensitivity. We suggest a daily screening maximum of 60 slides,
presuming six hours at the microscope. It seems essential to define such daily limits per
cytotechnologist and 24 hours. Definitions of the maximum slide numbers per year and
primary screener are not useful for quality assurance in daily practice.
Computer assisted microscopy: Computer assisted screening allows registration of screening
parameters, such as area coverage, screening time, stops, overlap of screen tracks and
screening meander. Regular recording of the screening pattern in the daily routine can
improve quality for an acceptable cost. (Is there evidence of improvement in accuracy by the
use of such systems, if not: drop it). Computer programs can ask the cytotechnologist to
rescreen all areas in a slide that might have been omitted. In future settings where cytological
diagnosis might be a co-operative approach inviting man and machine, the technical
microscope set up might need special microscope stages allowing for documentation of
screening coordinates and relocation of objects. Such microscope stages also allow presetting
of the screening meander.
3.1.4. Cell analysis: interpretation
The basic assumption of cytological diagnosis is that it is related to the histology of the
relevant tissue. This means that there is an equivalent appearance of cells even after the cells
80
are detached from tissue and all three-dimensional information is lost. This basis, including its
limitations, has been essential for cytology as a successful diagnostic tool.
There are two main approaches to the interpretation of cells that are usually combined. One is
based on the recognition and identification of cell types, the other is the criteria oriented
approach e.g. analysing cells for the so-called “criteria of malignancy”. A number of
characteristic cell types are important for the interpretation of cells. In most cases the
cytological diagnosis is based on a cell population rather than on a single cell. In any case
cytological interpretation is subjective. The complexity of these processes explains why
training (Chapter 10) and proficiency testing (Chapter 7) are so important in clinical cytology.
Separating "normal cellular findings" from "benign cellular changes" seems to be a major
problem. Since the days of Papanicolaou with his Pap classes I and II such separations have
survived. In the current Bethesda reporting system it is proposed to skip such a separation in
favour of a group that is called "negative for epithelial abnormality". Normal components of
cervical smears are squamous cells, endocervical cells and endometrial cells at certain age
groups and phases of the cycle. Atrophic epithelium may be considered as normal or in some
settings as a benign cellular change, especially if atrophy induces inflammation or
degeneration.
Patterns of different lesions are generally expressed by criteria catalogues. Knowledge of such
criteria lists is important for training as well as for practical diagnosis. Still, many cases do not
display the complete set of characteristics but show only some of them, possibly added to
some contradicting features.
3.1.5. Adequacy of the sample
Most report schemes nowadays have a component related to the adequacy of the sample. The
cytologist can answer the question, if the slide looks adequate for evaluation, but never will be
able to classify a smear as representative. Accepting the smear as representative is a clinical
decision based on the cytological outcome and the observations made by the smear taker at the
time of sampling. In general smears should contain both squamous epithelial cells and
endocervical columnar cells, which is documented in the report.
3.1.6. Cytological findings
Microscopic images of cervical cytological specimen can be found at: (Give the source: web
site address). Maybe a few images can be integrated in the text. To be discussed with Joe and
Ulrich.
Benign cellular changes
Numerous variants of benign cellular findings have been described. For many of them there is
no clinical relevance since they do not imply an increased risk for neoplasia. Rather many are
important if they are not interpreted correctly and lead to over-diagnosis.
Benign cellular changes may be related to: microbiological findings, inflammation; hormonal
influence/ atrophic squamous cells; ongoing or previous therapy; reaction to trauma; radiation;
intrauterine contraceptive device (IUD).
Glandular cells status post hysterectomy.
81
Clinical relevance of benign cellular changes: no clinical relevance at all; changes impeding repetition
of the smear; changes potentially inducing over-diagnosis (for instance follicular cervicitis, tissue
repair); findings needing patient treatment.
Cells indicating intraepithelial cell changes:
 Cells of mild dysplasia
 Cells of moderate dysplasia
 Cells of severe dysplasia and carcinoma in situ
 Atypical glandular cells
Cells from invasive cancer:
 Cells of invasive squamous cell cancer
 Adenocarcinoma of the endocervix
3.1.7. The cytological report
Cytologists or cytopathologists should use a standardised terminology for the reporting of
cytological findings. Results must be stored in a standard database format in a computer-based
laboratory information system (LIS). The LIS should use mnemonics or assigned codes for
rapid data entry. The LIS must allow for free-text capabilities which are necessary e.g.
for special interpretations, comments and recommendations that deviate from standard
routine. The LIS must allow easy transmission of information, communication of results,
programme management and evaluation.
Different report schemes are in use throughout Europe. Report forms are often used to
distribute medical and financial resources, and therefore difficult to change. Translation
among the different report schemes is highly desirable but not easy. Three principle criteria
should be applied when defining separate categories in a cytological reporting system: 1)
inter-rater reproducibility; 2) correlation with histological findings; 3) natural history.
Numerical report schemes like the Papanicolaou classification are obsolete but still common
in European laboratories and throughout the cytological literature. Annex 2a provides an
overview of reporting schemes currently used in the member states. In most European
countries a reporting system derived from the 1991 or 2001 version of the American Bethesda
System [Solomon, 2001] has been developed. Therefore a translation table proposing the
conversion of national terminology to the 2001 Bethesda system has been added (appendix
2b). Throughout time, several classifications were elaborated and will be elaborated, yielding
different splitting or lumping of cytological categories that compromise longitudinal statistical
analysis. In order to have some universal reference system for translation, a detailed
continuum of cytological entities has been described where cut-offs between previous and
future categories can be identified (appendix 2c).
3.1.8. Clinical applications of cervical cytology
Conventional cytology is still the standard method for primary cervical cancer screening.
Repetition of the Pap smear is used as triage method in case of minor cytological methods and
as follow-up method after treatment of lesions (see chapter 8).
82
3.2. Performance of cervical cytology
Programme sensitivity
Efficacy of conventional cytological screening for cervical cancer was never demonstrated in
randomised clinical trials (design type 1, see Table 1) but evidence of effectiveness is
nowadays widely accepted from observational studies (design types 2-4). For an overview of
the performance, we refer to the systematic review performed by the International Agency for
Research on Cancer in 1986 [Hakama, 1986]. Three- to five year screening in women 35-55
years old after 2 previous smears offered in an organised setting yields a reduction in
cumulative incidence of 91% to 84%. The programme sensitivity was lower and more
heterogeneous than in non-organised settings due to lower and more variable test sensitivity
(less rigorous quality control). More estimates of the relative protection (a performance
parameter directly related to programme sensitivity) offered by cytological screening, are
shown in tables x-y in chapter 2.
Cross-sectional test accuracy of cervical cytology
Problems of the identification of best gold standard for cervical cytology [Davey, --].
The cross-sectional diagnostic validity of cervical cytology for CIN using the histological
result of a biopsy, conus, endo-cervical curettage of hysterectomy as gold standard, was
evaluated in two meta-analyses [Fahey, 1995; McCrory, 1999 & Nanda, 2000]. We have
reanalysed data extracted from the most recent American meta-analysis. The results of the
meta-analytical pooling yielded estimates of accuracy that are summarised in Table 3.
Table 3
Table 3: Meta-analysis of test sensitivity and specificity of cervical cytology at 3 test thresholds (ASCUS+,
LSIL+ and HSIL+) for colposcopically or histologically confirmed presence of CIN2+ or CIN1+ (adapted from
McCrory, 1999) pooled from studies with complete and incomplete gold standard verification (adapted from
McCrory, 1999).
Table 3a: Outcome presence of CIN2+
All studies
Test threshold
Sensitivity (95% CI) # studies
ASCUS+
0.77
(0.64-0.90)
6
LSIL+
0.83
(0.80-0.90)
46
HSIL+
0.58
( 0.49-0.66)
45
Specificity
0.69
0.61
0.89
(95% CI)
( 0.57-0.82)
(0.55-0.67)
(0.87-0.90)
# studies
6
46
45
Only studies without verification bias
Test threshold
Sensitivity (95% CI) # studies
ASCUS+
0.65
(0.47-0.83)
1
LSIL+
0.77
(0.58-0.97)
6
HSIL+
0.87
( 0.78-0.96)
1
Specificity
0.85
0.92
1.00
(95% CI)
( 0.81-0.89)
(0.89-0.95)
(0.99-1.00)
# studies
1
6
1
83
Table 3b (continued): Outcome presence of CIN1+
All studies
Test threshold
Sensitivity
(95% CI)
# studies
ASCUS+
0.71
(0.67- 0.75)
32
LSIL+
0.67
(0.63-0.71)
72
HSIL+
0
Specificity
0.69
0.73
-
(95% CI)
(0.63- 0.75)
( 0.71-0.76)
-
# studies
31
72
0
Only studies without verification bias
Test threshold
Sensitivity
(95% CI)
ASCUS+
0.53
(0.22-0.84)
LSIL+
0.52
(0.38-0.66)
HSIL+
-
Specificity
0.97
0.96
-
(95% CI)
(0.94-0.99)
(0.94-0.98)
-
# studies
4
9
0
# studies
4
9
0
There is a tendency of higher sensitivity and lower specificity for CIN2+ than for CIN1+.
The sensitivity decreases and the specificity increases with higher cytological test threshold.
Studies without verification bias, where all subjects were submitted to the gold standard
showed higher sensitivity and lower specificity.
The low pooled sensitivity of cytology at threshold LSIL+ for presence of CIN1+, assessed in
studies with complete verification, being 52% (95% CI: 38-66%), has been used as a reason
for yearly as opposed to less frequent screening or as justification for newer more sensitive
methods. It must be remarked that the outcome was CIN1+. CIN1 represents a lesion that
usually regresses or, if progressive, has a high chance to be picked up while still non-invasive
at a subsequent screening. Studies included in the meta-analysis, a fortiori those with
complete gold standard verification, often concerned follow-up setting not representative for a
screening situation.
The test sensitivity of cytology for CIN (without precision of test and outcome thresholds),
estimated by modelling from the historical British Columbia cohort was 80% [Boyes, 1982;
Van Oortmarssen, 1991; Van Ballegooijen, 1997]. It concerned here sensitivity evaluated in
an organised screening setting with good quality control.
To conclude, it can be stated that the test sensitivity and specificity of the conventional Pap
smear are not known precisely. The sensitivity for CIN2+ at low cytological thresholds is
relatively high. The estimation of the accuracy varies by population characteristics (age,
screening history, screening or follow-ups situation) and study design properties (selection
bias, method of gold standard assessment, verification bias, independent assessment of gold
standard).
Nevertheless, convincing evidence is available with respect to the effectiveness of cytological
screening, if offered in a well organised setting with quality control at all levels.
84
4. Liquid based cytology (MarcARBYN, Ulrich SCHENCK, Odette REAL)
4.1. Description
Thin-layer cytology or liquid based cytology (LBC) is a new technique for transferring the
cellular material to the microscope slide. The cervical broom is usually recommended for
taking the sample. However a plastic extended tip spatula or the combined use of plastic
spatula and endo-cervical brush are also options.
The smear is not transferred in the usual way onto a slide (see appendix 1). The sampling
device carrying the material is immersed in a container with a special liquid transport
medium. The container is then sent to a specially equipped laboratory. At present, there are
several commercial systems available.
Several commercial systems have been developed in the last fifteen years, among which
ThinPrep (Cytyc, Boxborough, MA, US) and SurePath (formerly, AutoCyte PREP, TriPath
Imaging Inc., Burlington, NC, US) are the most well-known. With the ThinPrep-2000 or the
more fully automated ThinPrep-3000 processor the liquid is aspirated through a membrane
that detains the cellular material, which is then stamped onto a slide in the form of a very thin
layer, often called a monolayer. With the PrepStain machine the SurePath material is
sedimented through a density gradient [Howel, 1998]. Only ThinPrep and SurePath have so
far been approved in the United States by the American Food and Drug Administration
(FDA). Other fluid based systems are manufactured as wella.
Until recently, the FDA allowed only for ThinPrep to claim lower inadequacy rates and higher
detection rates of LSIL and HSIL in comparison with conventional cytology. For AutoCyte
only the label of improved preparation quality and equal detection of cytological abnormalities
was attributed. In May 2003, the FDA approved the claim of increased HSIL detection with
SurePath as well.
4.2. Rational for liquid based cytology
A first advantage of fluid based methods is that almost all the sampled cells are rinsed into the
liquid while with the conventional smear a selective portion of the cellular material may
remain trapped on the sampling device [Rubio, 1977]. Transfer via a fluid medium increases
the likelihood of representative smears [Hutchinson, 1993]. The ThinPrep and SurePath
systems produce circular areas that contain on average 50 000 to 75 000 randomly selected
cells, whereas the conventional Pap smear usually contains between 100 000 to 250 000 cells,
which is about one fifth of the cellular material available on the sampling device [Hutchinson,
1993].
Fixation of the cell material is optimal in LBC. However, the altered background requires
training and a period of adaptation for the cytologist [Austin, 1998]. Red blood cells and
mucus are for the most part absent and leukocytes are more evenly distributed. Epithelial
fragments, which are difficult to interpret on a classical smear, are for the most part
disaggregated during the preparation, while diagnostic clusters of columnar or metaplastic
a
Little information is available concerning other commercially available LBC systems such as: Cytoscreen®, Cyteasy®,
Labonord Easy Prep®, Cytoslide®, SpinThin®, PapSpin®.
85
cells are usually preserved. The microscopic visualisation of a calibrated thin layer of properly
distributed cells is more comfortable for cytological interpretation, which should facilitate the
evaluation of cytological structures [Linder, 1997; Austin, 1998].
Multiple smears can be made or additional investigations performed on the residual fluid (e.g.
DNA detection of the human papillomavirus or Chlamydia) without the necessity to recall the
woman concerned [Ferenczy, 1997; Sherman, 1997].
A thin-layer specimen might be a more proper target for automated screening devices.
A considerable disadvantage is the higher cost - both the capital investment and the operating
costs and the dependence on the manufacturer’s disposables.
4.3. Recent reviews, meta-analyses and pilot studies
Several reviews and meta-analyses regarding the performance of LBC were performed over
the last 5 years (see list of references). Conclusions formulated by the reviewing authors were
disparate and depended largely on selection criteria to include individual studies and the
considered performance parameters. Two types of study designs were distinguished: split
sample studies and direct-to-vial or two-cohort studiesa. Studies comparing detection rates for
low grade cytological abnormalities often yielded more favourable results for LBC, whereas in
studies focusing on accuracy for biopsy-confirmed high-grade CIN, no significant differences
between conventional and liquid-based cytology were found.
For this reason it was decided to conduct an exhaustive meta-analysis in the framework of the
evaluation of new screening methods in the European Union, which was one of the priorities
of the European Network for Cervical Cancer Screening. A complete overview of the results
can be found in a comprehensive report edited by the Scientific Institute of Public Health
[Arbyn, 2003]. This meta-analysis was more comprehensive than previous ones including all
studies documenting for LBC and CP: detection rates of cytological, accuracy relative to
colposcopically and/or histologically confirmed outcomes, quality judgement and screening
time as well as possible influencing factors.
4.3.1. Comparison of the accuracy of liquid-based cytology with the conventional
Pap-smear
Differences in detection in cytological abnormalities are shown in Table 4:
- Split sample studies
o More LSIL lesions are detected in LBC (liquid based cytology) than in CP
(conventional Pap smear): 14% (CI: 9-20%) more in ThinPrep slides and 32%
(CI: 11-55%) more in AutoCyte/SurePath slides. The detection rate ratios for
ASCUS and HSIL are not statically different from unity.
o The pooled inadequacy rate is lower in LBC preparations but this was
statistically not significant due to large-study heterogeneity.
a
Split sample studies: studies where a conventional Pap smear is prepared prior to rinsing the
residual material in a vial with liquid. Detection rates in conventional and liquid based smears
are compared.
86
-
Direct-to-vial studiesa
o More LSIL and HSIL lesions are detectable in LBC and this observation is
significant for both ThinPrep and AutoCyte/SurePath.
For ThinPrep: 72% (CI: 42-108%) more HSIL and 80% (CI: 52-112%) more
LSIL. For AutoCyte/SurePath: 47% (CI: 14-89%) more HSIL and 54% (CI:
25-90%) LSIL. Detection rates for ASCUS were similar.
o Detection rate ratios are considerably higher in direct-to-vial studies,
suggesting bias in split-sample studies in disadvantage of LBC. Spreading of
cellular material for a conventional Pap smear preparation might cause
selective removal of diagnostic elements that are not available anymore for
LBC.
Table 4: Meta-analysis: pooling of ratios of detection rates (LBC/Conventional Pap) [Arbyn, 2003].
Split sample studies
ThinPrep (beta, 2000 or 3000 version)
Test
Pooled
threshold
estimate
95% CI
# studies
Lower Upper
HSIL+
1.01
0.95
1.08
18
LSIL+
1.09
1.05
1.13
18
LSIL
1.14
1.09
1.20
18
ASC+
1.06
0.96
1.17
17
ASC
1.00
0.80
1.25
17
Direct-to-vial studies
ThinPrep
Test
Pooled
threshold
estimate
HSIL+
LSIL+
LSIL
ASC+
ASC
1.72
1.74
1.80
1.23
0.95
95% CI
# studies
Lower Upper
1.42
2.08
21
1.47
2.06
21
1.52
2.12
21
1.07
1.40
19
0.84
1.09
19
AutoCyte/SurePath (all versions)
Pooled
estimate
95% CI
# studies
Lower Upper
1.00
0.94
1.06
14
1.18
1.06
1.32
13
1.32
1.11
1.55
13
1.04
0.95
1.14
13
0.87
0.75
1.00
13
AutoCyte/SurePath
Pooled
estimate
95% CI
Lower Upper
1.47
1.14
1.89
1.52
1.24
1.86
1.54
1.25
1.90
1.19
0.96
1.46
0.93
0.67
1.31
# studies
7
7
7
7
7
Increased detection of cytological abnormalities (ratios >1) provides insufficient evidence for
improved sensitivity of LBC compared to CP. Therefore verification with a valid golden
standard is needed.
Differences in positive predictive value allow us to verify if higher cytological detection rates
in LBC are due to an increase in false-positive results. Positive predictive values for presence
of CIN2+ were pooled from studies with at least an 80% gold standard verification of testpositives. Pooled PPV ratios never were significantly different from unity. The relative
sensitivity for biopsy confirmed CIN2+, in split-sample studies with at least 80% verification
of positives, were again not significantly different from unity. It must be mentioned that
a
Direct-to-vial studies (or two-cohort studies): studies where all the collected cervical
material is transferred to the vial with liquid. Detection rates for LBC are compared with
series where conventional Pap smears were prepared.
87
numerous studies were excluded since they did not reach 80% of completeness in histological
verification of positive smears.
Verification of the true status of the cervical epithelium (presence or absence of CIN), by
colposcopy and biopsy should preferentially be performed on all subjects and should concern
essentially moderate or severe dysplasia or cancer. Verification of all study cases was only
performed in a few split-sample studies [Ferenczy, 1996a; 1996b; Bergeron, 2000; Confortini,
2002; Coste, 2003]. The results of these studies are summarised in Table 5.
Table 5: Ratio of sensitivity and specificity for CIN2+ of two liquid-based cytology systems (LBC) relative to
the conventional Pap smear (CP), pooled from studies with complete verification by colposcopy and/or biopsy
[Arbyn, 2003].
ThinPrep
AutoCyte/SurePath
95% CI
Test
threshold
HSIL+
LSIL+
ASCUS+
Ratio of
sensitivities
(LBC/CP) lower
0.95
0.88
0.97
0.92
0.99
0.96
95% CI
upper # studies
1.03
2
1.02
2
1.02
3
ThinPrep
Ratio of
Test
sensitivities
threshold (LBC/CP)
HSIL+
0.95
LSIL+
0.95
ASCUS+
0.96
upper # studies
1.11
1
1.05
1
1.02
1
AutoCyte/SurePath
95% CI
Test
threshold
HSIL+
LSIL+
ASCUS+
lower
0.81
0.86
0.91
Ratio of
specificities
(LBC/CP) lower
1.08
0.90
1.05
0.89
1.05
0.85
95% CI
upper # studies
1.30
2
1.24
2
1.30
3
Ratio of
Test
specificities
threshold (LBC/CP)
HSIL+
0.94
LSIL+
0.84
ASCUS+
0.92
lower
0.87
0.72
0.72
upper # studies
1.01
1
1.00
1
1.18
1
From the few completely verified datasets, no conclusion of improved accuracy of the LBC
systems for detection of histologically confirmed CIN2+ can be made. Coste [2003] even
concluded that the conventional Pap smear is superior to LBC, but this conclusion was based
on the outcome of CIN1+. Moreover, it must be stated that one of studies was done with the
beta-version of the ThinPrep system. All studies with complete verification were conducted
on split samples. Possibly some learning effects might have influenced negatively the results
for LBC since involved smear takers and cyto-technologists were not yet thoroughly
accustomed to the new technique. All these explanations are speculative.
4.3.2. Judgement of the adequacy of liquid-based cytology relative conventional Pap
smears
The proportion of inadequate smears and the proportion judged as “satisfactory but limited by
(SBLB)” and the reasons for limited quality judgement are shown in Table 6, where results are
summarised from split-sample and direct-to-vial studies for conventional Pap smears and the
ThinPrep and SurePath/AutoCyte systems.
There was no significant reduction in the pooled inadequacy rate observed in the split-sample
studies, whereas in direct-to-vial studies this reduction was more pronounced and significant
in SurePath/AutoCyte smears (rate ratio: 0.13; CI: 0.07-0.26). The lack of significance was in
88
general due to the large inter-study variability, which in turn can be attributed to poor
standardisation in definition and application of quality definitions. The same contrasts were
observed for the proportion of SBLB smears.
Most studies did not report complete quality information. The proportion of smears obscured
by blood or inflammatory material or inadequate fixation was reduced in liquid-based
cytology and this was significant in most situations. The proportion of inadequate smears due
to scanty cells was significantly higher in LBC with the exception of SurePath/AutoCyte in
direct-to-vial studies.
In split-sample studies LBC slides showed a significantly higher proportion of smears without
endocervical cells (EC-) compared to conventional smears. On the contrary, in direct-to-vial
studies, the percentage of EC- in LBC was similar (in case of ThinPrep) or lower (in case of
SurePath/AutoCyte) than in conventional cytology, confirming the disadvantage of LBC using
the split-sample study design. Keeping the sampling brush in the preservation liquid might
contribute to more optimal cellularity and presence of endocervical cells [Bigras, 2003].
Table 6: Frequency of quality judgement and distribution of reasons for limited quality of liquid and
conventional Pap (CP) smears; ratio of frequencies, pooled from split-sample and direct-to-vial studies [Arbyn,
2003].
Split-sample studies
Quality category
Unsatisfactory
Inadequate fixation
Scanty cells
Obscuration by blood
Inflammation
SBLB
ECObscuration by blood
Inflammation
Reasons of inadequacy
Unsatisfactory
Inadequate fixation
Scanty cells
Obscuration by blood
Inflammation
SBLB
ECObscuration by blood
Inflammation
#
ThinPrep
CP
Ratio: ThinPrep/CP
Frequency
95% CI
Frequency
95% CI
Estimate
95% CI
lower upper
lower upper
lower upper studies
1.4% 1.0% 1.8%
2.3% 1.6% 2.9%
0.73
0.39
1.39
19
0.0%
0.0% 0.0%
0.1% 0.0% 0.2%
0.24
0.06
1.02
5
1.5%
0.8% 2.1%
0.6% 0.3% 1.0%
2.03
1.60
2.57
6
0.2%
0.0% 0.3%
0.5% 0.1% 0.9%
0.39
0.26
0.59
5
0.0%
0.0% 0.1%
0.4% 0.0% 0.7%
0.13
0.07
0.25
4
14.1% 10.9% 17.4%
13.2% 10.3% 16.1%
0.3%
0.0% 0.6%
0.7%
0.1% 1.2%
18.1% 10.6% 25.5%
7.2% 5.0% 9.4%
3.6% 2.1% 5.1%
5.0% 1.4% 8.5%
0.87
1.89
0.08
0.16
0.62
1.49
0.02
0.07
1.23
2.39
0.26
0.39
7
8
6
6
#
AutoCyte/SurePath
CP
Ratio: AutoCyte/CP
Frequency
95% CI
Frequency
95% CI
Estimate
95% CI
lower upper
lower upper
lower upper studies
0.7% 0.5% 1.0%
1.5% 1.0% 1.9%
0.56
0.30
1.07
14
0.0%
0.0% 0.0%
1.2% 0.0% 2.4%
0.16
0.02
1.46
3
0.7%
0.0% 1.5%
0.5% 0.0% 1.2%
2.03
1.03
4.00
3
0.1%
0.0% 0.1%
0.5% 0.0% 1.2%
0.37
0.05
2.51
3
0.1%
0.0% 0.1%
1.9% 0.0% 4.1%
0.21
0.03
1.45
3
20.5% 13.2% 27.8%
20.3% 13.7% 27.0%
0.3%
0.0% 0.7%
1.5%
0.3% 2.8%
27.5% 17.0% 38.0%
16.1% 9.9% 22.3%
5.6% 0.4% 10.8%
8.6% 2.2% 14.9%
0.85
1.39
0.08
0.31
0.60
1.11
0.01
0.18
1.20
1.75
0.71
0.54
89
10
10
3
3
Table 7: (continued)
Direct-to-vial studies
Quality category
Unsatisfactory
Inadequate fixation
Scanty cells
Obscuration by blood
Inflammation
SBLB
ECObscuration by blood
Inflammation
Reasons of inadequacy
Unsatisfactory
Inadequate fixation
Scanty cells
Obscuration by blood
Inflammation
SBLB
ECObscuration by blood
Inflammation
Ratio: ThinPrep/CP
#
ThinPrep
CP
Frequency
95% CI
Frequency
95% CI
Estimate
95% CI
lower upper
lower upper
lower upper studies
1.0% 0.7% 1.3%
1.9% 1.3% 2.5%
0.70
0.39
1.27
16
0.0%
0.0% 0.0%
0.0% 0.0% 0.1%
0.11
0.01
2.43
1
0.9%
0.1% 1.7%
0.5% 0.0% 1.5%
2.95
0.06 139.28
2
0.4%
0.0% 1.1%
0.1% 0.0% 0.3%
1.31
0.02 113.36
2
0.1%
0.0% 0.1%
0.2% -0.2% 0.6%
0.14
0.07
0.29
2
14.8%
7.2%
0.1%
0.5%
5.9% 23.7%
5.4% 9.1%
0.0% 0.1%
0.1% 0.8%
19.6% 15.8% 23.4%
7.6% 4.3% 10.9%
1.2% 0.1% 2.4%
6.2% 2.2% 10.2%
0.51
1.17
0.09
0.12
0.23
0.76
0.01
0.07
1.11
1.82
0.55
0.21
12
5
3
4
#
AutoCyte/SurePath
CP
Ratio: AutoCyte/CP
Frequency
95% CI
Frequency
95% CI
Estimate
95% CI
lower upper
lower upper
lower upper studies
0.3% 0.2% 0.4%
2.7% 1.6% 3.9%
0.13
0.07
0.26
8
0.0%
0.0% 0.0%
0.4% 0.3% 0.5%
0.00
0.00
0.07
1
0.2%
0.0% 0.5%
1.1% 0.6% 1.6%
0.09
0.01
1.41
2
0.0%
0.0% 0.0%
0.0% 0.0% 0.1%
0.38
0.10
1.41
1
0.0%
0.0% 0.0%
0.0% 0.0% 0.0%
0.13
0.09
0.18
1
7.0%
3.7%
0.8%
0.7%
5.5%
0.4%
0.0%
0.0%
8.5%
7.0%
2.3%
1.7%
15.8%
12.2%
1.3%
5.9%
7.5% 24.0%
7.3% 17.2%
0.0% 3.8%
0.0% 15.6%
0.39
0.52
0.24
0.13
0.26
0.20
0.03
0.09
0.60
1.33
2.11
0.18
4.3.3. Demonstration pilot projects conducted in Scotland and England
In a pilot study, conducted by the Scottish Cervical Screening Programme, [SCSP, 2002],
smear takers were randomised in two groups, collecting respectively conventional Pap smears
and ThinPrep preparations. Smears and vials were sent to 4 laboratories where cytotechnologists were previously trained to interprete LBC smears. Results are summarised in
Table 7.
The positive predictive values (cut-offs not documented) were similar for both preparation
techniquesa. Subsequent to the publication of these results, the Scottish Government decided
to convert completely to liquid based cytology.
a
The cytological- & histological thresholds used to compute PPV were not documented in the
SCSP report [2002].
90
5
3
2
2
Table 7: Proportion of inadequate smears and cytological abnormalities and ratios observed in two randomised
groups of women from 4 Scottish areas. In group-1, a ThinPrep smear, and in group-2, a conventional smear was
taken [SCSP, 2002].
Cytological category
Unsatisfactory
Borderline
Mild dyskaryosis
Moderate dyskaryosis
Severe dyskaryosis
Total (N=30288)
Proportion
ThinPrep
CP
1.86%
8.00%
3.67%
4.35%
2.10%
1.09%
0.97%
0.48%
1.09%
0.59%
Ratioa
(TP/CP)
0.23
0.84
1.93
2.01
1.84
(95% CI)
(0.20-0.26)
(0.76-0.94)
(1.60-2.32)
(1.52-2.66)
(1.42-2.38)
The NHS demonstration project (England)
As part of a 12-month pilot project conducted in 3 selected laboratories, LBC was introduced,
after a learning transition period of 3 to 6 months [Moss, 2003]. At the same time, triage of
women with results showing borderline abnormality or low-grade dyskaryosis with HPV
DNA was incorporated as well. The cytological results of the first six months of the pilot
period (which are not influenced by the HPV triage) were compared with the four previous
years where exclusively conventional cytology was used. In two laboratories, the ThinPrep
system was introduced and in another the SurePath. It must be mentioned that different
sampling devices were use before and after introduction of LBC.
The proportion of inadequate smears before and after conversion to LBC is shown in Table 8.
The inadequacy rate dropped significantly in all laboratories and in all age groups after
introduction of LBC. The quality of conventional smears increased with age. No age
differential was observed anymore in the LBC period. The inadequacy rate was lowest in the
SurePath laboratory.
Table 8. Prevalence of inadequate smears by preparation system. (computed over the 4 CP years and the 1 st six
months of the LBC period) [adapted from Moss, 2003].
Preparation system
Conventional Pap
ThinPrep
SurePath
% inadequate
95% CI
9.7
2.0
0.9
9.4-10.0
1.8-2.2
0.8-1.1
The relative changes in the detection of rates of cytological abnormalities in LBC versus CP
by age group and by laboratory are shown in Table 9.
In the ThinPrep laboratories significantly more SIL and HSIL lesions, and in one of them (lab
C) also more borderline lesions, were found. In the laboratory where SurePath was used, less
HSIL and borderline lesions were detected. The excess of lesions detected with LBC was
concentrated in the age-group 20-34 years.
a
Confidence intervals are approximate. In the SCSP report it was stated that in half of the
women a CP was taken and in the other half a TP. We assumed exactly 15 144 individuals in
both groups. This allowed us to calculate confidence intervals.
91
Table 9. Relative change in the prevalence of abnormal smears, SIL and HSIL in LBC smears in comparison
with conventional Pap smears (CP), by laboratory, by age (crude and weighted) [adapted from Moss, 2003].
Laboratory
A
B
C
Age
20-34
35-49
50-64
20-64 (mha)
Abnormal (>=borderline)
RR
95% CI
0.99
0.94-1.04
0.94
0.90-0.99
1.37
1.30-1.44
RR
1.15
0.96
0.93
1.06
95% CI
1.11-1.19
0.91-1.02
0.85-1.01
1.03-1.09
SIL (>=mild dyskaryosis
RR
95% CI
1.25
1.19-1.31
1.00
0.93-1.08
1.73
1.60-1.86
RR
1.25
1.03
1.03
1.18
95% CI
1.19-1.31
0.94-1.13
0.87-1.23
1.13-1.23
HSIL (>=mod. dyskaryosis
RR
95% CI
1.10
0.98-1.24
0.85
0.75-0.96
1.55
1.36-1.76
RR
1.18
1.01
0.75
1.10
95% CI
1.08-1.28
0.87-1.16
0.55-1.01
1.02-1.18
4.3.4. Influencing factors
With the exception of test- and gold standard thresholds no other factors could be retained as
influencing significantly the accuracy measures. The assessed covariates were: composition
of the study population, clinical setting (screening, follow-up or mixed), the version of the
LBC-system (betaTP, TP2000, TP3000; CytoRich, AutoCyte PREP, SurePath, other LBC
systems), collection devices, training of smear takers and readers, blinding of screeners,
reviewers, colposcopists, and histologists, quality control of cytotechnologists' 1st diagnosis,
definition and completeness of golden standard verification, thresholds for cytology and
histology, length of follow-up period, and last but not the disclosed interests of the researcher
and involvement of the manufacturers of devices. The lack of significance might be due to
poor reporting and insufficient information compromising the power of multi-variate analyses.
4.3.5. Economical aspects of liquid based cytology
In 10 studies included in the aforementioned meta-analysis screening time was measured. The
simple mean was 229 and 239 seconds respectively for thin-layer and conventional Pap
smears (reduction of nearly one third) [Arbyn, 2003]. The need for adjusting the
microscope’s objective is eliminated in LBC since the cellular material is localised in only one
very thin layer [Payne, 2000]. McGoogan [1996] reported that interpretation of thin layer
specimen was more tiring. Papillo remarked that the shorter evaluation time was off-set partly
by a longer processing/preparation time [Papillo, 1992]. This remark is not valid anymore for
the more automated processors. In the UK demonstration project, introduction of LBC was
considered cost-effective essentially due to economical reasons: reduction in the proportion of
inadequate smears and subsequent decline in repeat smearsb and the shorter interpretation
time. Subsequent to the publication of the results, the National Health Service decided to
convert to LBC as a preferred cervical cancer screening method (NICE, 2003).
4.3.6. Training, time trend effects
The introduction of LBC requires that cytotechnicians be adequately trained. Often in
literature the first months of experience with LBC are documented, when cytologists are still
a
(mh): overall Mantel-Haenzel adjusted relative risk.
It must be remarked that the economic savings brought about by the reduction in
unsatisfactory smears is expected to be substantially lower in other EU member states, since
the inadequacy rate is substantially lower in the rest of Europe.
b
92
not sufficiently accustomed to the new technique (McGoogan, personal communication). A
learning effect was demonstrated in the NHS demonstration trial, with higher abnormality rates
during the first two months of the pilots [Moss, 2003].
4.4. Propositions for future research
Future research should apply more rigid controlled study designs: randomised trials
(conventional versus liquid based methods), in settings representative of a screening situation,
with clear definitions of study outcomes including validation of test results by acceptable gold
standards and provide detailed information on potentially influencing factors. Difference in
detection rates of histologically confirmed CIN2+ or CIN3+ seems an acceptable intermediate
study outcome. Studies assessing the adequacy of slides should imply all components of the
quality judgement: cellularity, composition, preservation, aspect, and presence of obscuring
elements. Such trials were recently set up in the Netherlands and Italy.
Costs and effects and cost-effectiveness ratios (cost per additional case of CIN2+ detected)
should be extended towards more relevant outcomes such as costs per avoided case of cancer
or life years saved, based on the best available precise local financial information, accurate
estimates of effects derived from trials and updated meta-analyses and plausible assumptions
on the natural history of cervical cancer.
4.5. Conclusions
The meta-analysis confirms the hypothesis that the split-sample study design creates a bias
that defavorises LBC. In split-sample studies with complete colposocpy/histology verification
no difference in sensitivity and specificity was found considering the outcome of CIN2 or
more serious disease. However, two-cohort studies show substantial increases in detection
rates of LSIL and HSIL in liquid-based smears whereas the positive predictive value for
moderate dysplasia of more severe disease is not reduced significantly in comparison with
conventional Pap smears. From this observation a gain in sensitivity without loss in
specificity can be assumed. The level of evidence for this deduction is rather low. So we
have to conclude that insufficient high-quality data are currently available to judge definitively
on the superiority of fluid based cervical cytology.
The quality of LBC smears has improved considerably. The inadequacy rate is lowest in
SurePath preparations.
Interpretation of LBC smears requires less time.
While awaiting definitive results from well-conducted RCTs, we can recommend both types
of preparations, conventional and liquid-based cytology with a certain reservation for the costeffectiveness for LBC, which depends on local situations.
93
5. Automated cytological screening (Pekka NIEMINEN, Ulrich
SCHENCK, Marc ARBYN)
5.1. Description of automated screening devices
Two commercial systems were extensively studied in the 1990s: PAPNET (Neuromedical
Systems Inc. (NSI), Suffern, New York, USA) and the AUTOPAP System (Neopath Inc.,
Redmond, Washington, USA).
PAPNET includes neural network software and traditional imaging technology. It selects 128
of the most suspect fields in conventional Pap smears and presents this on a video screen.
A cytologist interpretes the images on the screen and decides to carry out manual screening
when abnormalities are recognised or suspected
The Food and Drug Administration (FDA) of the United States has approved PAPNET for
quality control of slides interpreted as negative after conventional screening. NSI has recently
been declared bankrupt. TriPath Imaging Inc. (formerly AutoCyte, Burlington, NC, US) has
acquired the intellectual property of PAPNET.
AUTOPAP is a computerised scanning device designed for algorithmic classification of
conventional Pap smears. It designates a score based on the likelihood that the slide contains
an abnormality. AUTOPAP selects a predetermined proportion of slides that need further
manual screening. The FDA has approved AUTOPAP for quality control and for primary
screening [Dunton, 2000].
In the meantime, newer devices are emerging targeting liquid based cytology smears, for
instance: FOCAL POINT (TriPath Imaging Inc.) and IMMAGER (Cytyc, Boxborough, MA,
US).
5.2. Rational for automated screening
Automation assisted screening is aimed to increase sensitivity and specificity by finding e.g.,
small atypical cells, known to be very difficult to find in conventional screening. These
include both squamous and glandular cells. The performance of screening is designed to
increase by excluding part of the slides from manual screening or by enriching the most
atypical cells to images or to be studied by the microscope. By enhancing the effectiveness of
the screening work, automation is thought to allow more slides to be screened without
changing the number of staff. This would be an advantage, because in many countries there is
a severe shortage of cytotechnicians. As part of these automated devices are capable at
processing either conventional or liquid based smears, it allows them to be used in different
kinds of screening programmes.
The aims for automated screening are: (1) to increase sensitivity and specificity of cytological
screening; (2) to decrease the workload of cytotechnicians and cytopathologists; (3) to
decrease the cost of the screening programmes; (4) finally, to decrease the incidence and
mortality of cervical cancer.
94
5.3. Evaluation of the performance
Several articles have been published concerning the performance of automation-assisted
screening [Bartels, 1998; Bergeron, 2000; Doorneward, 1999; Duggan 2000; Fahey, 1995;
Halford, 1999; Kok, 1996, 2000; Koss, 1997; Michelow, 1997; Prismatic Trial Team, 1999;
Wilbur 1996]. They show generally a better test sensitivity with at least the same specificity
as conventional screening. Most of these articles have been retrospective (quality control)
and/or involved relatively small numbers of smears. The Prismatic study [Prismatic Trial
Team, 1999], showed also equal sensitivity but better specificity for automated screening as
well as better productivity (faster screening) in a prospective study with 21 700 smears. Only
two randomised, prospective public health trials in a primary screening setting have been
published thus far. First of these studies reported clearly higher detection rates of CIN3 or a
more serious finding, particularly, of in situ-carcinoma and invasive carcinoma [Kok, 1996].
The second study, in an organised screening setting and involving several cytological
laboratories, did not clearly confirm the result [Nieminen, 2003] (Table 10). Instead, the latter
study showed almost equal sensitivity when compared with the traditional cytological
screening technique. Specificity and positive predictive value were at an equal level in
automation-assisted screening when compared to manual conventional screening. These
parameters were reported only in the latter study (Table 11 and Table 12).
Table 10. The number (N) and proportion (/1000) of screenees by histology and study arm. Odds ratios (OR)
with the 95% confidence intervals (CI) of histologically verified cervical lesions in the Papnet arm in comparison
with the conventional screening arm (logistic regression) (Kok, 1996: table A; Nieminen, 2003: table B).
A
Histology
Papnet arm
Total 65527
Conventional arm
Total 25767
OR
p
N
/1000
N
/1000
Invasive cancer
42
0.64
8
0.31
2.16
P < 0.05
In situ carcinoma
79
1.20
18
0,68
1.76
P < 0.05
CIN3
124
1.89
44
1,70
1.11
N.S
OR
CI
B
Histology
Papnet arm
Conventional arm
Total 36,225
Total 72,461
N
/1000
N
/1000
Invasive cancer
3
0.08
4
0.06
1.50
0.30-6.80
CIN 3
51
1.4
100
1.4
1.02
0.72-1.42
CIN2
51
1.4
104
1.4
0.98
0.70-1.36
CIN1
40
1.1
96
1.3
0.83
0.57-1.20
36,080
996
72,157
996
1.00
reference
Normal and other
95
Table 11. Specificity of the Papnet and conventional Pap-smear test with cut off levels of ASCUS+ and LSIL+
for invasive cancer and for an outcome of CIN2+ or invasive in primary screening setting. Total N=108205
women [Nieminen, 2003]
Negative histology
Negative Pap smear
Specificity %
Papnet
36,222
33,447
92.3
Conventional
72,453
67,241
92.8
Papnet
36,171
33,447
92.5
Conventional
72,353
67,240
92.9
Papnet
36,222
35,972
99.3
Conventional
72,453
71,890
99.2
Papnet
36,171
35,970
99.4
Conventional
72,353
71,887
99.4
Cytological threshold: ASCUS+
Outcome: invasive cancer
Outcome: CIN2+
Cytological threshold: LSIL+
Outcome: invasive cancer
Outcome: CIN2+
Table 12: Positive predictive values (PPV, %) of Papnet and of the conventional cytology, and the difference in
the PPV (Papnet vs. conventional) with its p-value for cytology at threshold LSIL+ and ASCUS+, by various
histologically confirmed outcomes in a primary screening setting. Total N=108205 women [Nieminen, 2003].
Cytology threshold LSIL+
Histological
outcome
Papnet
N=252
PPV (N)
Invasive cancer
1.2
Difference
Papnet vs.
conventional
(p-value)
Papnet
N=2,777
PPV (N)
0.5 (p=0.49)
0.1
CIN3+
20.6 (52)
17.9 (101)
2.8 (p=0.35)
CIN2+
40.1 (101)
35.2 (199)
CIN1+
55.2 (139)
50.8 (287)
(3)
Conventional
N=565
PPV (N)
Cytology threshold ASCUS+
0.7
(4)
(3)
Conventional
N=5,214
PPV (N)
0.1
Difference
Papnet vs.
conventional
(p-value)
(4)
-0.0 (p=0.65)
1.9 (54)
2.0 (103)
-0.0 (p=0.92)
4.9 (p=0.18)
3.8 (105)
4.0 (207)
-0.2 (p=0.68)
4.4 (p=0.25)
5.2 (145)
5.8 (303)
-0.6 (p=0.28)
96
The technological development is very rapid in this branch. New technology is emerging and
some of the older devices are no longer commercially available.
When implementing the new methods, it is necessary to carefully ascertain and evaluate the
performance of the method in primary (public health) screening up to the final invasive end
points with randomised prospective studies. Thus it is important to organise the trials in such
a way that the technology studied can be used for several years in the trial, irrespective of its
current or future commercial availability [Nieminen, 2003].
Conclusion
The few randomised prospective studies and also the other performance studies have shown
that automation assisted screening may be feasible as a part of routine primary screening and
it seems to perform at least equally well compared to conventional organised well working
screening programme with devices tested. Automation assisted screening can eventually
improve the results of a suboptimal screening organisation, but it may be very difficult to beat
the results of a well organised, high quality screening. However, it seems to be possible to do
more samples with same quality.
Presently there is a quiet moment in the field of automation assisted screening because of nonmedical reasons, but in the near future new generation of screening devices are launched to
the market. These new models should also be tested in prospective randomised trials before
adopting them in to the routine screening.
97
6. Colposcopy (Joe JORDAN, Jean-Jacques BALDAUF, Daniel DA SILVA,
Marc ARBYN)
6.1. Description
The colposcope is a type of loop, which allows observation of the cervix and vagina, under
optimal illumination at magnification between x6 and x40. The aim of colposcopy is to allow
the trained colposcopist to identify a premalignant disease of the cervix.
In a premenopausal woman, examination can be carried out at any phase of the menstrual
cycle, but it is optimally performed during the estrogenic phase. In patients with atrophic
cervical epithelium or cervico-vaginitis, examination should be carried out after prior
estrogenic preparation or appropriate antiseptic treatment. It is better not to perform
colposcopy after pelvic examination or after a cervical smear has been taken. Scraping of the
cervix may cause mucosal erosions or bleeding and hinder examination of the endocervix.
After examination of the vulva, an appropriate speculum is inserted taking due care not to
injure the cervix. Examination should be started at a low magnification after rinsing the cervix
with normal saline and removing any excess cervical mucus. The green filter may be used at
this stage of the examination to observe the vascular patterns.
Prior to colposcopy, a 3 or 5 % acetic acid solution is applied to the cervix. An acetowhite
reaction occurs when the squamous epithelium is abnormal. Acetic acid causes tissue oedema
and superficial coagulation of intracellular proteins, thus reducing the transparency of the
epithelium. Unfortunately, not all areas of acetowhite epithelium indicate the presence of
premalignant disease: for example, areas of immature metaplasia are densely aceto-white.
A further technique, is to apply Schiller’s iodine or lugol solution. Normal squamous
epithelium is rich in glycogen and stains dark brown with iodine, whereas premalignant
squamous epithelium is deficient in glycogen and is non-staining. It is a good method for
demarcating abnormal areas when the cervix is actually being treated. Again, not all nonstaining areas represent premalignant disease.
Complete colposcopic examination requires observation of the original squamous epithelium,
the entire transformation zone, the squamocolumnar junction and as much of the columnar
epithelium of the cervix as possible. Locating the squamocolumnar junction is a key
procedure in colposcopic interpretation. If the squamocolumnar junction is not or only
partially visible, i.e. if the internal - endocervical - limit of the normal or atypical squamous
epithelium is not entirely apparent, then the colposcopy is unsatisfactory.
Colposcopy requires long-term experience to acquire an expertise in colposcopic pattern
recognition. The expert colposcopist may be able to predict the histological diagnosis quite
accurately but in general the colpo-histological correlation is only moderate. Even in several
years of colposcopic practice, the inter-observer and intra-observer variations in colposcopic
interpretations may not reach Kappa values greater than 0.50. The specificity of colposcopy is
low, due to over-interpretation of aceto-white areas in the transformation zone. The
sensitivity is with regard to endocervical lesions. Micro-invasive carcinoma can be
misinterpreted as well.
98
Once the colposcopic examination is completed, it is essential that all observations be entered
on a structured colposcopy chart. The chart should show the situation of the squamocolumnar
junction and clearly define the topography and nature of the different lesions, as well as
biopsy sites (see chapter 2 and chapter 8).
6.2. Indications for colposcopy
Colposcopy is not intended to be a screening tool because of its low specificity.
In case of cytological abnormalities, colposcopy with directed biopsies, cone biopsy and or
endocervical curettage should be performed before any form of treatment is given n order to
confirm the cervical lesion and determine its severity, its topography and its extension.
Colposcopy is an essential triage method that orients diagnostic exploration. For a more
extensive description of indications and best practice guidelines please refer to chapter 8.
99
7. HPV DNA detection (Joakim DILLNER, Pär SPAREN, Marc ARBYN)
7.1. Human papillomavirus infections
7.1.1. Introduction
Human papillomavirus (HPV) is the most common sexually transmitted infection. Over 90
different types of HPV have been identified and fully sequenced, while over 120 putative
types exist that have been partially characterized [zur Hausen 1999; zur Hausen 2000]. All
HPV types are epitheliotropic, completing the growth cycle only in differentiating
keratinocytes of the skin and the anogenital and oropharyngeal mucosa. Since the specific host
cell receptor(s) for HPV have not been determined, it is not known if the strict tropism is
determined at the receptor level or is determined by host factors required for replication
[Evander, Frazer et al. 1997]. Approximately 35 HPV types are known to infect the human
genitalia, causing a range of clinical states including asymptomatic infection, genital warts,
cytologic abnormalities of the cervix, and invasive cervical cancer [zur Hausen 1999].
HPV types are assigned numerical designations once the DNA sequence has been established
and a comparison to previously known types have found less than 90% homology in the L1,
E6 and E7 regions of the virus [de Villiers 1999]. Isolates with more than 90% homology to
known HPV types are classified as subtypes. The different HPV types are subdivided into two
categories: “high risk” and “low risk, originally based on whether the HPV type could or
could not be found in cervical cancer or CINIII specimens. The HR-HPV types show
phylogenetic relatedness. Individuals infected with low risk viruses have a very low risk for
developing of cervical cancer. The low risk types such as HPV 6 and HPV 11 are associated
with benign, hyperproliferative lesions, commonly referred to as genital warts, or condyloma
acuminata [Gissmann, Wolnik et al. 1983]. High risk types such as HPV types 16, 18, and
others cause dysplastic lesions of the cervix, including invasive cancer.
The causative relationship between HPV and cervical carcinoma has provided the incentive to
target HPV in cervical cancer prevention efforts. This chapter will deal both with the use of
HPV testing in cervical cancer screening, as well as the prospects for development of a safe
and effective prophylactic vaccine and the impact it would be predicted to have on cervical
cancer screening programs.
Rate and determinants of acquisition of genital tract HPV infection
The epidemiological studies described below were mainly performed using cervical
specimens from young women. These studies indicate that HPV infection is characterized by
a very high rate of acquisition. A two-year follow-up of previously uninfected female
university students in the United States found a cumulative incidence of HPV infection (any
type) of 32.3% [Winer, Lee et al. 2003]. The most important determinant of HPV acquisition
is a change of sexual partner, where each change of sexual partner involves a substantial risk
for HPV infection. In Sweden, the risk for seroconversion to HPV 16 increases linearly
approximately 4% for each life-time sexual partner up to a plateau of about 32% among
women with an average of eight lifetime sexual partners [Dillner, Kallings et al. 1996]. A
longitudinal cohort study of teenage girls found that without sexual experience, none became
seropositive for HPV 16 or HPV 33, whereas among girls who had had five or more partners,
54% were positive at some point in time [Andersson-Ellstrom, Dillner et al. 1994]
[Andersson-Ellström, Dillner et al. 1996].
An interesting determinant of acquisition is how long the partners have known each other
before having sex, with risks for HPV acquisition being substantially higher if sex occurs with
100
a partner known for less than eight months [Winer, Lee et al. 2003]. This may be related to
the fact that HPV infection usually has a self-limited course (see below). Having a male
partner reporting a high life-time number of sexual partners or other concomitant partners is
also a risk factor for cervical cancer [Winer, Lee et al. 2003]. Finally, lack of circumcision
appears to promote penile HPV infections that do not clear and become persistent which also
would tend to favor HPV transmission [Castellsague, Bosch et al. 2002].
Condom use has shown protection against HPV infection in several studies [Karlsson,
Jonsson et al. 1995] [Bleeker, Hogewoning et al. 2003; Hogewoning, Bleeker et al. 2003], but
many studies have failed to detect a significant protection. Several reasons for the
inconsistent protection have been proposed, including incorrect or inconsistent use, and the
fact that the HPV infection may exist in a wider area of the genital epithelium than is covered
by the condom [Winer, Lee et al. 2003].
HPV infection in virginal women has in several studies been found to be rare or non-existent
[Krüger Kjaer, Chackerian et al. 2001] The very low prevalence of HPV infection among
virginal women has been associated with non-penetrative sexual contact [Winer, Lee et al.
2003]. Neonatal HPV infection has been the subject of much discussion. Several studies
have reported that HPV DNA can be commonly detected among children born to HPVinfected mothers. Since HPV seropositivity is very rare among pre-adolescents [af
Geijersstam, Eklund et al. 1999; Plummer M and Franceschi 2002], it would appear that
neonatal HPV DNA contamination does not usually lead to infection. An extensive study that
also included an investigation of whether the children were seroconverted for HPV (a classical
criterion for infection), did document that neonatal HPV infection can occur, although it was
quite rare [Manns, Strickler et al. 1999].
Rate of clearance and determinants of clearance/persistence.
HPV infection is characterized by a very high rate of spontaneous clearance. Several cohort
studies of young HPV DNA positive women have been quite consistent in their estimates of a
70% clearance rate during a 12-month follow-up [Hildesheim, Schiffman et al. 1994;
Evander, Edlund et al. 1995; Ho, Bierman et al. 1998]. After 18 months, more than 80% of
infections have cleared [Ho, Bierman et al. 1998] and a five year follow-up study found a
clearance rate of 90% [Elfgren, Kalantari et al. 2000]. Clearance rates are lower in older
women. HPV-positive women of approximately 35 years of age were followed for 19 months
on average, and only 55% cleared their initial infection.
Women with persistent type-specific positivity are at increased risk to develop invasive
cervical cancer [Nonnenmacher, Hubbert et al. 1995; Remmink, Walboomers et al. 1995;
Wallin, Wiklund et al. 1999]. Women with type-specific HPV persistence who had normal
Pap smears have been commonly found to have an undiagnosed high-grade CIN (in about
29% of cases) [Elfgren 2003]. The determinants of HPV persistence and clearance are
therefore also key determinants of cervical cancer risk. Several studies have found that HPV
16 (the most commonly detected type in cervical cancers) has a higher propensity to persist
than other HPV types do [Hildesheim, Schiffman et al. 1994; Brisson, Bairati et al. 1996; Ho,
Bierman et al. 1998; Moscicki, Shiboski et al. 1998; Elfgren, Jacobs et al. 2002].
7.1.2. HPV in relation to other risk factors for cervical cancer
HPV infection is by far the most significant risk factor for cervical cancer. Relative risks
usually exceed 100 in case control studies and are usually in the range of 10 to 30 in
prospective studies [Lehtinen, Luukkaala et al. 2001; Clifford, Smith et al. 2003]. The
attributable proportion (fraction of cervical cancers predicted to disappear if the risk factor
101
was eliminated) is in excess of 50% even when only counting HPV 16. If both HPV 16 and
HPV 18 were eliminated, almost 70% of cervical cancers would be prevented. If the four of
the most common oncogenic HPV types (HPV types 16, 18, 31 and 45) were eliminated, a
protective effect of about 80% is predicted.
Systematic monitoring of which HPV types actually cause cervical cancer in various parts of
the world is important in order to design of HPV vaccines and to predict the effect of
vaccination in different populations. The systematic world-wide review by Clifford et al,
gives a good overview of the type distribution in invasive cervical cancers. The most
common HPV types were, in order of decreasing prevalence, HPV16, 18, 45, 31, 33, 58, 52,
35, 59, 56, 6, 51, 68, 39, 82, 73, 66 and 70. For squamous cell cervical cancer the most
common type was HPV 16, followed by HPV 18, whereas for adenocarcinoma the order
between these two types was reversed. However, there are some deviations from the typical
distribution of HPV types, e.g. HPV types 52 and 58 have been reported to be quite common
in studies from Taiwan, China, Japan, Mozambique [Castellsague, Menendez et al. 2001] and
Costa Rica [Wang, Schiffman et al. 2003]. There is an interesting overrepresentation of three
HPV types in invasive cancer series as compared to high-grade CIN series, namely HPV types
16, 18 and 45, suggesting that these three types are not only able to cause high-grade CIN, but
that high-grade CIN caused by these types may have a greater risk to progress to cervical
cancer [Clifford, Smith et al. 2003]
Most of the other known risk factors for cervical cancer are the same as those that determine
either HPV acquisition or persistence, with some exceptions, as described below. Studies of
smoking as a determinant of HPV acquisition or persistence have been inconsistent.
However, smoking has consistently been associated with cervical cancer. The issue of whether
this could be due to residual confounding by HPV has been repeatedly discussed, as smoking
habits correlate with sexual behavior in several populations. A dose-response effect is seen
and randomized intervention studies of smoking cessation among women with low grade CIN
have shown a beneficial effect [Cuzick, Szarewski et al. 1995]. At present, the bulk of the
evidence indicates that smoking may indeed be a cervical cancer risk factor. If so, it probably
acts at the CIN progression stage in cervical carcinogenesis.
Oral contraceptive pills (OCPs) have been studied as a risk factor for cervical cancer. A large
series of studies over a number of years have shown inconsistent associations with cervical
cancer. OCP use is of course intimately associated with sexual habits, which may lead to
confounding in some populations. However, recent systematic reviews of the literature have
indicated that OCP use with duration of more than 5 years is a cervical cancer risk factor
[Smith, Green et al. 2003].
Multiparity is consistently detected as a risk factor in populations where a substantial
percentage of the women have multiple children [Schiffman and Brinton 1995]. The
mechanism is unclear, with hormonal effects and/or repeated trauma to the cervix being the
most commonly proposed explanations.
A series of early cross-sectional case-control studies found a strong association between
infection with herpes simplex virus (HSV) and cervical cancer [Aurelian 1984]. The case for
HSV as a risk factor became weaker when several prospective cohort studies failed to find an
association [Vonka, Kanka et al. 1984; Lehtinen, Hakama et al. 1992]. Recently, a large
cross-sectional case-control study found an association between HSV and cervical cancer, but
recent prospective studies have found no association. Prospective studies are less prone to
selection biases and, perhaps more importantly in this case, they do not have the problem of
reversed causality. HSV is a common virus that persists in a latent form in the body and may
be reactivated by a serious disease such as cancer. It is therefore plausible that the association
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seen selectively in studies based on samples taken after cancer diagnosis may be due to the
cancer causing a virus reactivation rather than a virus being a risk factor for the cancer.
Infection with Chlamydia trachomatis has been repeatedly found to be associated with
cervical neoplasia and invasive cancer in cross-sectional case control studies. This association
has commonly been thought to be attributable to confounding by HPV. During recent years,
an association with Chlamydia has also been a consistent finding of several biobank-based
prospective studies with invasive cervical cancer as endpoint. In a large population-based
cohort study of HPV-positive women, that examined most of the proposed risk factors for
HPV persistence, history of Chlamydia infection was the only significant risk factor for HPV
persistence [Silins, Tedeschi et al. 2002]. In the studies of cervical cancer it is noteworthy
that C. trachomatis consistently associated with squamous cell carcinoma of the cervix, but
not cervical adenocarcinoma or other anogenital cancers such as vulvar or vaginal cancer,
even though these also have HPV and sexual behavior as a risk factor [Koskela, Anttila et al.
2000; Anttila, Saikku et al. 2001; Smith, Munoz et al. 2002]. More work is needed to clarify
the true role of Chlamydia infection in cervical cancer.
7.1.3. Immunogenetic factors: an increased risk for cervical cancer?
Lastly, immunogenetic factors have been implicated in cervical carcinogenesis, with class II
HLA haplotypes being the most well studied ones. Although there is a substantial
heterogeneity between studies [Konya and Dillner 2001; Daling, Madeleine et al. 2002],
DQw3 and DR15/DQ6 are the ones that have shown most consistent associations. DR15/DQ6
increases the risk for cervical cancer in particular among HPV 16-positive subjects, suggesting
a specific interaction with HPV 16 epitopes rather than a more general immunoregulartory
phenomenon as explanation. The association with DR15/DQ6 has in particular been found in
a series of studies from different groups in various Scandinavian countries [Sanjeevi,
Hjelmdahl et al. 1996; Helland, Olsen et al. 1998; Beskow, Josefsson et al. 2001; Ghaderi,
Wallin et al. 2002]. It is not entirely clear if the association is more pronounced in
Scandinavia or if it has been more readily detected owing to the Scandinavian tradition of
minimizing selection bias by performing population-based studies. Similar tendencies have
also been reported in other populations, notably in New Mexico and the U.K. [Glew, DugganKeen et al. 1993; Apple, Becker et al. 1995]. DR15/DQ6 has been associated with both HPV
16 infections becoming persistent rather than clearing, CIN, and cervical cancer in
longitudinal studies [Beskow, Josefsson et al. 2001]
7.2. HPV tests: principles and laboratory practises
7.2.1. Quality control criteria
Reproducibility: The early literature on epidemiology of HPV based on HPV DNA testing
was inconsistent. Careful validation of technologies subsequently showed that some of the
early assays commonly gave misclassified results. Even a moderate amount of
misclassification in HPV testing can lead to severe underestimations of relative risks. This has
been most clearly pointed out by Schiffman and Schatzkin [Schiffman and Schatzkin 1994]
who found that 2 essentially similar studies performed in the same laboratory, one study with
moderately reproducible technology, the other with carefully validated PCR technology
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resulted in completely different conclusions: Estimations of the relative risk for CIN in case of
HPV positivity of 2.3 or >10, respectively.
Although HPV DNA tests have improved greatly in recent years, clinicians and investigators
should remember the possibility that positivity may not always reflect a true infection. Blinded
reanalysis of a panel containing the same set of samples, on 2 different occasions, is a simple
and readily doable method to assess the current standards of testing.
Sensitivity and specificity: Assessing the sensitivity and specificity of HPV tests is not
straightforward, as these measures are dependent on knowledge of results of a “Gold standard
test” that should reflect the truth. For clinical practice evaluation purposes, it is also more
useful to consider test performances in relation to the desired properties of the test, rather than
in relation to some form of laboratory standards. In this context the desired property of the test
is that testing should reduce the risk of cervical cancer.
There are two major sides of this desired property: 1. Negative women should have a low risk
of developing cervical cancer. The duration of this low risk determines testing frequency and
general cost-efficiency of a screening program. 2. Positive women should have a high risk of
developing cervical cancer. For positive women there should be a treatment and surveillance
option that reduces their risk for cervical cancer.
These basic criteria are more complicated than they may seem at a first glance. Whereas it is
abundantly clear from a large amount of studies that HPV-negative women are at a very low
risk to have high-grade CIN or cancer at the time they are tested, much less data exists
regarding the duration of this low risk. A 1997 modeling study that assumed the duration of
low risk to last only 1 year found that HPV screening was not advantageous over presently
used programs, but would be both more effective and cost-effective if the low risk lasted for
10 years (41). Today, there exists a substantial amount of longitudinal evidence that has found
that HPV-testing has indeed a long-term predictive value for future occurrence of high-grade
CIN or cervical cancer [Lorincz and Richart 2003].
None of these longitudinal studies has compared different HPV tests to determine which one
has the best desirable test characteristics in providing a long-term protective effect against
cervical cancer, but it can be concluded that both one of the general primer PCR test systems
(GP5+/GP6+) and the Hybrid Capture II HPV test (see below) do confer at least some longterm protection.
7.2.2. Types of HPV tests
Based on these considerations, there are three major HPV DNA tests that could be
contemplated for use in screening:
1. Hybrid Capture II is a commercially available HPV test which is based on RNA-DNA
hybridization and sensitive detection of the formed DNA/RNA hybrids, but which is not
based on target DNA amplification.
This test has the obvious advantage of availability in a standardized kit format that can be
used by most laboratories. The test has been used in several longitudinal studies and has
been shown to have sensitivity for detection of high-grade CIN and cancer that is
considered to be useful. A disadvantage is that the test does not provide the possibility to
determine the HPV type in the sample. There is only an answer for presence or absence of
oncogenic HPV, as the test hybridizes with a mixture of probes for the oncogenic HPV
types. Apart from the HPV types that the test is designed to detect, the test has also been
found to detect additional HPV types that cross-hybridise with the probe mix (47).
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2. General primer PCR based on the primer pair GP5+/GP6+.
This test amplifies a 140 bp region in the L1 gene of papillomaviruses and has shown an
amazing specificity and sensitivity for prediction of high-grade CIN. The test has been
developed to a simple, rapid enzyme immunoassay-PCR (EIA-PCR) format that is
suitable for processing very large amounts of samples. An international validation study
that was performed before the start of a primary HPV screening trial in Sweden found
limited interlaboratory variation (Kappa statistics of at worst 0.88, at best 1.0) .
Comparison of reproducibility between different HPV tests in the same study found
comparatively low agreement, implying that intermethod variability is considerably
greater than intramethod interlaboratory variation.
HPV typing of positive samples can be accomplished by several methods. The most
commonly used method is reverse hybridization, originally reported by Forslund et al,
1994, which hybridizes the labeled PCR products with HPV genomes or probes
immobilized on membranes. Comparison of reproducibility of different HPV tests for
determining the exact HPV type in the sample found unacceptably low agreements. At
present, it cannot be investigated with certainty which HPV types that it is cost-effective
to screen for, because meta-analyses of literature using different HPV typing methods
can not be performed.
3. General primer MY09/11 system
This PCR test amplifies a 450 bp region in the L1 gene. The test is presently used with
an improved primer design (PGMY09/11 primers), that has been found to have better
consistency and better sensitivity for a broad range of HPV types than the original
MY09/11 primers (42).
There are several methodological studies that have compared this test to either the
Hybrid Capture or the GP5+/GP6+ PCR system. The sensitivity for detection of cervical
neoplasia appears to be about the same, but there is a disturbing amount of discrepant
results. Qu et al found an overall agreement of 0.79 (kappa statistic) (48) and Elfgren et
al reported a kappa statistic of 0.68 when comparing MY09/11 and GP5+/GP6+ (49).
Peyton et al found a kappa of 0.58 when comparing MY09/11 and Hybrid Capture (50).
Part of the discrepancies, but only part, can be explained by differential sensitivities for
certain HPV types (e.g. the MY09/11 primers are less sensitive for amplification of
HPV 35 and GP5+/GP6+ are less sensitive for amplifying HPV 53 and 61.
There is also a striking difference in the amount of samples that are simultaneously
positive for several HPV types by the different systems, with MY09/11 assays reporting
much more multiple HPV positivities (48). It is not clear whether the difference is
attributable to a reduced ability of the GP5+/GP6+ to amplify multiple infections or
whether there are inherent problems with the subsequent HPV typing in the MY09/11
system.
4. SPF10 is an ultrasensitive short fragment PCR from the L1 region. [Kleter, van Doorn
et al. 1998; Kleter, van Doorn et al. 1999]. Whereas studies using this method have
found similar proportions of women with current or future cervical lesions to be
positive, there is a higher proportion of cytologically normal women, who do not
develop CIN on follow-up, that test positive using this system (Review by Snijders,
2003), resulting in lower relative risks and positive predictive values. As discussed
above, high analytical sensitivity in terms of ability to detect low amounts of virus
105
does not necessarily increase the sensitivity of the test for screening purposes, as
detection of a very low viral load is not associated with an increased risk for future
CIN [Josefsson, Magnusson et al. 2000; Ylitalo, Sorensen et al. 2000].
In summary, although HPV testing in general has advanced enormously in performance, the
major available tests that are conceivable for use in primary HPV screening have been
evaluated to somewhat different extents as desirable screening tests. It is clear that continuous
quality control monitoring is necessary for all studies and clinical uses and that further
improvements to reduce discrepancies would be desirable. With regard to the use of general
primer PCR-based tests, the fact that they are not yet commercially available is currently a
significant drawback.
7.3. Clinical applications of HPV testing
7.3.1. Use of HPV testing in primary screening
Assessments on the effects of various possible actions to increase the effectiveness of cervical
cancer prevention have shown that the most effective action is to ensure a high attendance rate
of the population. The second most effective action is ensuring that women with cancer or
precursor lesions who do attend will not have a false negative diagnosis.
Ninety-five percent or more of cervical cancers and high-grade CIN lesions are HPV-positive,
and HPV DNA testing is well established to have an increased sensitivity, compared to the
Pap smear, for detection of prevalent high-grade CIN. Apart from the promise of an increased
sensitivity for high-grade CIN, the fact that most adenocarcinomas are HPV-positive holds
promise for possible improvement of cervical screening programs, as cytological screening
has limited effect against cervical adenocarcinoma.
The major question regarding the use of HPV DNA testing is whether the advantage of
increased sensitivity results in an overall improvement considering the increased costs and,
usually, lower specificity of HPV testing.
These guidelines are based on identification of the most recent systematic review of the
literature. In addition, the information in the databases of Europe against Cancer-sponsored
trials of HPV testing have been considered.
In the systematic review by Lörincz and Richart, trials of HPV DNA testing were compared.
They concluded that HPV DNA testing was a more sensitive indicator for prevalent highgrade CIN than either conventional or liquid cytology. A combination of HPV DNA and
Papanicolaou testing had almost 100% sensitivity and negative predictive value. The
specificity of the combined tests was slightly lower than the specificity of the Papanicolaou
test alone, but this decrease could potentially be off-set by greater protection from neoplastic
progression and cost savings available from extended screening intervals. One "doublenegative" HPV DNA and Papanicolaou test indicated better prognostic assurance against risk
of future CIN 3 than 3 subsequent negative conventional Papanicolaou tests.
The basic performance indicators (Sensitivity, specificity, positive and negative predictive
value) of HPV DNA testing and Pap smear are shown below (modified from Lörincz and
Richart, with permission).
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Figure 1. Sensitivity of Papanicolaou, HPV DNA, and Papanicolaou and HPV DNA, for CIN 2/3 (London,
Reims, Newfoundland, Cape Town [individual tests only; the combined Papanicolaou and HPV DNA tests in this
series refer to CIN 3], Shanxi [individual tests only; the combined Papanicolaou and HPV DNA tests in this
series refer to CIN 3]) or CIN 3 (Seattle, Guanacaste, Cape Town [both Papanicolaou and HPV DNA tests; the
individual tests in this series refer to CIN 2/3], Shanxi [both Papanicolaou and HPV DNA tests; the individual
tests in this series refer to CIN 2/3], Hannover, Morelos). Data are for women aged 30 years and older, except for
the study from Newfoundland. Combined Papanicolaou and HPV DNA test data were unavailable for the London
and Seattle studies.
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I find this approach to early if you say earlier that more studies have to be done.I would say
that many algoritm evaluations are presently undrway
Figure 2. Specificity of Papanicolaou, HPV DNA, and Papanicolaou and HPV DNA, for CIN 2/3 (London,
Reims, Newfoundland, Cape Town [individual tests only; the combined Papanicolaou and HPV DNA tests in this
series refer to CIN 3], Shanxi [individual tests only; the combined Papanicolaou and HPV DNA tests in this
series refer to CIN 3]) or CIN 3 (Seattle, Guanacaste, Cape Town [both Papanicolaou and HPV DNA tests; the
individual tests in this series refer to CIN 2/3], Shanxi [both Papanicolaou and , HPV DNA tests; the individual
tests in this series refer to CIN 2/3], Hannover, Morelos). Data are for women aged 30 years and older, except for
the study from Newfoundland. Combined Papanicolaou and HPV DNA test data were unavailable for the
London, Seattle, and Reims studies.
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Zielinski et al J.Pathol 2001
Figure 3. Positive predictive value of Papanicolaou, HPV DNA, and Papanicolaou and HPV DNA, for CIN 2/3
(London, Reims, Newfoundland, Cape Town [individual tests only; the combined Papanicolaou and HPV DNA
tests in this series refer to CIN 3], Shanxi [individual tests only; the combined Papanicolaou and HPV DNA tests
in this series refer to CIN 3]) or CIN 3 (Seattle, Guanacaste, Cape Town [both Papanicolaou and HPV DNA
tests; the individual tests in this series refer to CIN 2/3], Shanxi [both Papanicolaou and HPV DNA tests; the
individual tests in this series refer to CIN 2/3], Hannover, Morelos). Data are for women aged 30 years and older,
except for the study from Newfoundland. Combined Papanicolaou and HPV DNA test data were unavailable for
the London, Seattle, and Reims studies.
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Figure 4. Negative predictive value of Papanicolaou, HPV DNA, and Papanicolaou and HPV DNA, for CIN 2/3
(London, Reims, Newfoundland, Cape Town [individual tests only; the combined Papanicolaou and HPV DNA
tests in this series refer to CIN 3], Shanxi [individual tests only; the combined Papanicolaou and HPV DNA tests
in this series refer to CIN 3]) or CIN 3 (Seattle, Guanacaste, Cape Town [both Papanicolaou and HPV DNA
tests; the individual tests in this series refer to CIN 2/3], Shanxi [both Papanicolaou and HPV DNA tests; the
individual tests in this series refer to CIN 2/3], Hannover, Morelos). Data are for women aged 30 years and older,
except for the study from Newfoundland. Combined Papanicolaou and HPV DNA test data were unavailable for
the London and Seattle studies.
AGE GROUPS TO BE TARGETED AND SCREENING INTERVALS FOR HPV
SCREENING PROGRAMS.
The greatest cost-benefit of ordinary Pap smear screening programs is when a country goes
from no screening at all to one life-time cytological test. This single test at the optimal age,
about 35 years of age, is estimated to give 25% of the protective effect of an entire screening
program with smears every third year. By analogy, the greatest relative benefit of HPVscreening is expected when a country goes from no HPV testing to one life-time HPV test.
The time point of this life-time test should be sufficiently late in life such that most HPV
exposures have already occurred, but sufficiently early that the risk that invasive cervical
cancer has already developed is low.
Several large studies have evaluated the prevalence of HPV infection in various age groups.
Although HPV prevalence is high in younger age groups, this prevalence declines markedly
with increasing age. This may be attributed to the fact that most infections are cleared
spontaneously and changes in sexual behavior by age, resulting in decreasing rates of
acquisition. By 35 years of age, the prevalence of infection with oncogenic HPV types is 1-8%
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[Cuzick, Szarewski et al. 1995; Hagmar, Kalantari et al. 1995; Chua, Wiklund et al. 1996;
Rozendaal, Walboomers et al. 1996; Kjellberg, Wiklund et al. 1998; Cuzick, Beverly et al.
1999; Forslund, Antonsson et al. 2000]. The strongly increasing age-specific incidence in the
ages shortly before 40 years of age, imply that the optimal age to start HPV screening has to
be after 30 years of age, but before 40 years of age.
The Europe against Cancer-sponsored trial in Sweden of nationwide population-based HPV
screening of 12.500 35-year old Swedish women found a joint prevalence for the 14 most
common oncogenic HPV types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 or 68)
of 7.3%.
Evident conclusions are that:
i)
cost-efficiency of HPV testing will be poor in age groups <30 years of age, because
HPV is more common and cervical cancer is rare;
ii)
HPV prevalences may vary between different countries and between regions within
countries (e.g. between urban and rural regions) resulting in that cost-efficiencies
may vary between populations. There is some evidence for differences between
populations in the systematic reviews of the literature on predictive values of HPV
testing;
iii)
to be helpful in the planning and evaluation of the potential usefulness of HPV
screening programs, HPV-epidemiological studies should be population-based,
large-scale, use established reference methods for HPV testing and target age
groups where HPV screening is conceivable (i.e. 30-60 years of age).
RECOMMENDATIONS ON AGE GROUPS AND SCREENING INTERVALS FOR HPV
TESTING:
 Research is needed to determine the age-specific epidemiology of HPV infections in
different populations.
 Cost-efficiency modelling studies need to be repeated in different populations, that may
differ in associated costs, rates of HPV infection of different types as well as in
background rates of other risk factors for cervical cancer.
POSSIBLE STRATEGIES TO IMPROVE THE SPECIFICITY OF HPV TESTING
FOR PRIMARY SCREENING
Longitudinal studies of HPV infections show that the infections normally clear within 1-2
years [Hildesheim, Schiffman et al. 1994; van Doornum, Prins et al. 1994; Evander, Edlund et
al. 1995; Ho, Burk et al. 1995]. In a population-based study of 611 women 19-25 years old,
20.3% were positive for HPV DNA at enrollment. However, these same 276 women were also
tested 2 years later, and only 2 women tested positive for the same oncogenic HPV type as 2
years earlier [Evander, Edlund et al. 1995].
The high rate of clearance of HPV DNA on follow-up is a consistent finding also in carefully
controlled studies using extremely sensitive detection methods (nested PCR)(3). However, it
is clear that the women who develop CIN or cancer are persistently positive for HPV DNA in
repeated tests [Remmink, Walboomers et al. 1995; Chua and Hjerpe 1996; Rozendaal,
Walboomers et al. 1996; Cuzick, Sasieni et al. 1999].
Simply repeating the HPV test to identify if the woman is persistently infected or not is
therefore one possible method to increase the specificity of primary HPV screening for
identifying the women who are truly at risk of developing cervical cancer. Two HPV tests
with a 1-year interval will, among 35-year-old women, identify about 50% of initially HPVpositive women as being persistently positive [Forslund, Antonsson et al. 2000]. The
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clearance rate among 35-year-old women is somewhat lower than among adolescents,
presumably because of an accumulation of persistent infections with time.
Another possible method to identify the women at highest risk is by testing for a high amount
of viral DNA, “high viral load” [Clavel, Masure et al. 1999; Josefsson, Magnusson et al.
2000; Ylitalo, Sorensen et al. 2000]. Contamination of samples at the clinic or at the
laboratory is a notorious problem in PCR assays. However, most contaminated samples
contain very low levels of virus. Restricting analyses to samples found to contain substantial
amount of virus therefore increases the probability that a positive result does indeed reflect a
true infection and thereby increases the predictive value of the test.
HPV serology is another method that is also known to be able to predict the risk of invasive
cervical cancer up to 15 years before diagnosis [Dillner, Lehtinen et al. 1997] and is also
associated with progression to CIN 3 among HPV DNA positive women [Bontkes, de Gruijl
et al. 1999]. For a comprehensive literature review on HPV antibodies, the reader is referred
to [Dillner 1999].
Other risk factors that have been found to increase the risk that HPV-positive women develop
CIN or cervical cancer are also conceivable screening targets. The HLA haplotypes DQw3 and
DR15/DQ6 increase the risk for both CIN and cervical cancer, as determined by metaanalyses of the literature of this subject [Konya and Dillner 2001]. Smoking is consistently
reported as a risk factor for invasive cervical cancer, but not as a risk factor for HPV
persistence. Indeed, some studies even find an inverse association with HPV persistence [Ho,
Burk et al. 1995]. Presumably smoking works only at the later stages of cervical
carcinogenesis. Assessing infections with Chlamydia trachomatis may also be a method to
predict increased risk of cervical cancer among HPV DNA positive women [Koskela, Anttila
et al. 2000]. Finally, testing for HPV integration appears to increase the predictive value that
the HPV positive sample is derived from a sample that contains CIN or cervical cancer [Klaes,
Woerner et al. 1999].
The possibility of using HPV testing for primary screening and the Pap smear for triaging
among HPV-positive women has also been proposed [Lorincz and Richart 2003].
RECOMMENDATIONS ON TRIAGING AMONG HPV POSITIVE WOMEN IN
PRIMARY SCREENING
 Repeated HPV testing has been established as improving specificity without significant
loss of sensitivity (HPV persistence is established as being a necessary risk factor for
HPV-associated cancers), but has not yet been widely evaluated in practise or in costefficiency models.
 Several methods for triaging among HPV positive methods have been proposed. Further
research on their usefulness is warranted. Such research should be guided by costefficiency modelling studies to better assess which demands on the performance indicators
that are required in order for a triaging test to be useful
FOLLOW-UP AND LONGITUDINAL PERFORMANCE
In screening programs in general, the longitudinal performance indicators are the most
relevant ones, as they determine the duration of the protective affect and thus the length of the
screening interval and the cost-efficiency of the entire program.
Evidence is available from several studies that have followed HPV DNA positive women for
up to 10 years. It is consistently found that the positive predictive value of HPV testing is
considerably increased, if the risk to have a high-grade CIN during the next 10 years is
considered (not merely the risk to have a high-grade CIN at the time point of diagnosis).
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These observations form the basis for proposals to use the HPV test in screening programs
with a lengthened screening interval compared to programs based on Pap smear screening
only.
However, the fact that the HPV test has a substantial longitudinal predictive value for future
development of high-grade CIN also presents problems.
To label a previously healthy woman as a high-risk individual for cancer development,
requiring repeated tests at frequent intervals, is a considerable psychological stress. The small
size of the group of women requiring follow-up and a duration of intensive follow-up after
testing HPV positive that is not unreasonably long is necessary for primary HPV screening to
be a realistic option.
If the status of a healthy HPV-positive woman as a high-risk patient for cancer were
permanent, the negative effect of primary HPV screening and associated costs would be
considerably greater than if the HPV positivity cleared.
However, several studies have now investigated whether conventional treatment (conization)
of HPV-positive CIN also results in a clearance of the infection. The answer is unambiguously
yes [Bollen, Tjong-A-Hung et al. 1996; Elfgren, Bistoletti et al. 1996; Bollen, Tjong-A-Hung
et al. 1997; Chua and Hjerpe 1997; Strand, Wilander et al. 1997; Kjellberg, Wadell et al.
2000; Nobbenhuis, Meijer et al. 2001]. Whether the infection is still present after treatment
predicts risk of recurrence of their CIN lesions [Chua and Hjerpe 1997] (Paraskevaidis). These
results indicate that follow-up will not be necessary for life, but only until HPV-positive
women become HPV-negative. It is currently unknown whether it is possible to augment
clearance of HPV infection (in the absence of cytological abnormalities) by some form of
treatment. With the current status of our knowledge the clinician would need to monitor the
HPV-positive patients either until cervical lesions develop (These can then be treated by
currently established treatment protocols) or until the women become HPV-negative.
RECOMMENDATIONS:
 Further research is necessary to better define the longitudinal performance indicators
(sensitivity, specificity and positive and negative predictive values) of HPV DNA testing
as well as of combined HPV DNA testing and cytology.
 Research into new methods of specific treatment of HPVinfection would be important to
minimise the time HPV-positive women need to be followed-up with intensified
screening.
 Research into optimal follow-up algorithms of HPV-positive women is necessary.
USING COST-EFFICACY MODELLING TO DESIGN HPV SCREENING
PROGRAMS.
In a pioneering series of studies by van Ballegooijen et al, mathematical modelling has been
used to assess both what program designs are likely to be most cost-efficient as well as to
identify critical areas of uncertainty where research is needed most (van Ballegooijen). Major
conclusions that could be drawn from these studies were that the longitudinal performance of
the HPV test was critical for achieving cost-efficiency, as the cost and lower specificity of
HPV screening needs to be compensated by a longer screening interval in order to be costeffective.
The Europe against Cancer-sponsored trial in Sweden has included a component with
mathematical modelling of cost-efficacy. The fact that the Swedish trial was population-based
and that population-based registry data is available since over 30 years of organised screening
results in the possibility to provide reliable input data to the model. As emphasized above,
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critical input data may differ between populations and it is not certain that the data can be
generalised outside Sweden. As the study is not yet published, it is summarised here:
Two alternative primary cervical cancer-screening policies were examined. Routine
cytological screening was compared with HPV DNA testing as an adjunct to cytology in
women over 32 years of age. The aim was to detect or rule out persistent infections with highrisk HPV types and by that improving screening-test performance. This approach could
increase sensitivity to detect high-grade cervical lesions (CIN2/3, cancer in situ) and at the
same time reduce costs by increasing the screening interval for those women who have
negative combined test results. In accordance with recent WHO guidelines, a comparison of
cytology mass screening from 32 to 60 years of age was also compared to no screening.
Compared with routine cytology screening, one life-time HPV DNA test with a PCR method
at 35 years of age as an adjunct to existing 3-yearly cytological screening increased costs and
the number of CIN2/3 detected without significant changes in life expectancy or number of
detected cervical cancers. However, if combined cytology and HPV testing was performed
three times at ages 32, 41 and ended at age 50, costs were the same as for 3-yearly cytology
screening, but the life expectancy increased and the number of expected life-time cases of
invasive cervical cancer was reduced from 26 to 17 in a modelled cohort of 10.000 women.
Reducing the costs of HPV testing in a sensitivity analysis with 33% did not substantially
change the cost-effectiveness results. Compared with no screening, a mass-screening program
with 3-yearly cytology from age 32 did actually save more health care resources than it
utilized (because of the reduction in cervical cancer treatment costs), with a considerable
reduction in the number of detected invasive cervical cancers.
The results are in line with modelling studies performed in the Netherlands and the United
Kingdom. Regarding the cost savings by the screening program, the results are in line with
data from another Nordic country (Finland) that also has organised screening and has
comparable levels of medical costs.
RECOMMENDATION:
Design of HPV screening trials, screening policies and/or policy evaluation studies should be
based on cost-efficiency modelling studies, specific to each population to be targeted for
screening.
PRIMARY HPV SCREENING IN GENERAL: What types of studies are necessary and what
should their endpoint be?
1. Setting: Experiences of disorganized cytological screening clearly imply that organized
screening systems are superior for reduction of cervical cancer incidence. If the future goal
is to find new technologies that could be used in organized, population-based call and
recall screening systems, this aspect should be considered already at the evaluation stage as
it will be valid to estimate how various policies would work (if implemented) only if the
trials are performed nested within such programs.
2. Desirable Study Design: Although the epidemiological evidence shows that the cervical
cancer protective effect of cervical screening is very strong [Ponte´n, Adami et al. 1995],
the fact that cervical screening was not evaluated by randomized trials before
implementation has delayed the rational implementation of cervical screening program and
resulted in long-standing debates of what the efficacy of the screening really is [Raffle,
Alden et al. 1995].
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An obvious lesson from history is to not repeat the mistake of introducing a new primary
screening program without first performing randomized trials to determine its effect at the
population level.
However, the demand for randomized studies has been increasingly challenged during recent
years. Randomized studies are very costly, resulting in overwhelmingly large amounts of
research funds being consumed, particularly if late endpoints such as mortality are desired.
They are also slow, providing results only after many years. The health benefits are delayed
and the trial results may have become outdated when they finally are obtained, as testing
technologies are improved all the time. In the HPV field, the improvement of HPV tests over
time is a telling tale. There are also new potential cervical cancer screening markers being
developed based on entirely different principles, e.g. the detection of the cell cycle regulatory
protein p16INK [Klaes, Friedrich et al. 2001].
The most compelling argument against randomized trials is that their effects may not be
generalizable, as the high quality setting of trials run by dedicated scientists is different from
the setting that will be used in a public health care policy [Hakama, Elovainio et al. 1991].
The mathematical modeling study has therefore been proposed as an alternative that will
provide answers in a timely fashion [Royston 1999]. While there is no arguing that the
modeling studies in the HPV screening field have provided valuable information on the
potential benefits of HPV screening, it is disturbing to note how different modeling studies
produce substantially different results [Cuzick, Sasieni et al. 1999; Sherlaw Johnson, Gallivan
et al. 1999; Myers, McCrory et al. 2000]. Part of the discrepancies, but only part, is due to the
use of different estimates of the input variables, most notably regarding cost, progression and
regression rates, sensitivity and specificity of tests. Input variables are usually estimated from
the scientific literature, which varies enormously in quality and the setting of studies.
Another alternative type of evaluation is the randomized health care policy, which means that
the new policy is not introduced in general but only for some regions or some birth cohorts. In
this strategy, research funds are not required and results do apply to a real health care policy,
not merely to the research setting. This approach has been successfully applied e.g. in the
Finnish mammography program [Hakama, Elovainio et al. 1991].
RECOMMENDATION:
A judicious use of a combination of randomised trials, modelling studies and randomised
health care policies is suggested. Modeling should be used to investigate optimal settings and
study designs for reduction into practice in randomized trials with intermediate endpoints
(such as protection against high-grade cervical intraepithelial neoplasia). Effects on
intermediate endpoints can then be used in further modeling studies to estimate effects on late
endpoints such as mortality and/or to design randomized health care policies.
7.3.2. Use of HPV testing in HPV triaging of equivocal smears
The most recent systematic review is the Europe Against Cancer-sponsored meta-analysis by
Arbyn et al (2003) [Arbyn, et al. 2003]. The conclusion is that available evidence indicates an
improved accuracy of HPV testing, using the Hybrid Capture-II assay, in comparison with the
repeat-Pap smear for the detection of high-grade intraepithelial neoplasia of the uterine cervix
among women with equivocal cytological results.
RECOMMENDATION:
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Well-validated HPV testing for detection of high-grade intraepithelial neoplasia of the uterine
cervix among women with equivocal cytological results in organised screening can be
considered. (For a detailed presentation see Chapter 8 in Guidelines).
7.3.3. Use of HPV testing in follow-up post treatment of CIN
The most current systematic review (Paraskevaidis, Arbyn, 2004, in press) is the basis for
these guidelines.
The standard treatment for CIN is different types of cervical surgical procedures.
Excision is effective and enables histopathological examination of the resulting cone biopsy
contrary to cryotherapy and laser vaporization. Evaluation of the excision margin status does
not reliably predict residual or persistent disease [Jansen, Trimbos et al. 1994; Soutter, de
Barros Lopes et al. 1997]. Recurrent CIN is reported in 0.3-23% of women with free cone
margins and in 6.9-84.8% of women without free margins [Dobbs, Asmussen et al. 2000;
Paraskevaidis, Lolis et al. 2000; Gonzalez, Zahn et al. 2001; Paraskevaidis, Koliopoulos et al.
2001]. Follow-up using Pap-smears after treatment is still the most common
procedure[Jansen, Trimbos et al. 1994] but has been questioned because of relatively low and
variable sensitivity for CIN detection [Martin-Hirsch, Koliopoulos et al. 2002]. Because HPV
infection is an essential cause of CIN, HPV DNA detection may be preferable as a rapidly
occurring intermediate endpoint for monitoring treatment efficacy. Several studies have found
that HPV DNA is commonly cleared after effective treatment for CIN and that persistence of
HPV DNA predicts recurrence[Elfgren, Bistoletti et al. 1996; Chua and Hjerpe 1997;
Kanamori, Kigawa et al. 1998; Mann, Steele et al. 1998]. These studies have used several
different treatment modalities which were found to have varying success in clearance of HPV
DNA, suggesting that HPV DNA testing is useful for evaluation of different treatment
modalities.
The meta-analysis of Paraskevaidis, Arbyn et al identified 11 studies on evaluating the use of
HPV testing after conservative treatment for CIN [Elfgren, Bistoletti et al. 1996; Chua and
Hjerpe 1997; Distefano, Picconi et al. 1998; Bollen, Tjong et al. 1999; Kjellberg, Wadell et al.
2000; Nagai, Maehama et al. 2000; Kucera, Sliutz et al. 2001; Lin, Tseng et al. 2001;
Nobbenhuis, Meijer et al. 2001; Paraskevaidis, Koliopoulos et al. 2001; Acladious, Sutton et
al. 2002]. Eight studies [Elfgren, Bistoletti et al. 1996; Distefano, Picconi et al. 1998; Bollen,
Tjong et al. 1999; Kjellberg, Wadell et al. 2000; Nagai, Maehama et al. 2000; Kucera, Sliutz
et al. 2001; Lin, Tseng et al. 2001; Nobbenhuis, Meijer et al. 2001; Acladious, Sutton et al.
2002] were prospective, and three studies [Chua and Hjerpe 1997; Paraskevaidis, Koliopoulos
et al. 2001]were retrospective. The total number of women included in these studies were 900,
of whom 678 (75.3%) were considered as receiving a successful treatment, whereas 222
(24.7%) were considered treatment failures.
There was marked heterogeneity in several aspects of the included studies. The grade of
intraepithelial lesion differed both within and between studies. In most studies, the initial
sample included different grades of cervical intraepithelial neoplasia, while 3 studies [Chua
and Hjerpe 1997; Nagai, Maehama et al. 2000; Lin, Tseng et al. 2001] included only women
with high-grade/CIN III lesions. Excisional methods of treatment (various conization
methods) were used in all studies, while destructive methods (e.g. cryocoagulation and
LASER evaporation) were also employed in three studies[Bollen, Tjong et al. 1999; Kjellberg,
Wadell et al. 2000; Acladious, Sutton et al. 2002]. Polymerase chain reaction (PCR) methods
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were employed in 8 studies [Elfgren, Bistoletti et al. 1996; Chua and Hjerpe 1997; Distefano,
Picconi et al. 1998; Bollen, Tjong et al. 1999; Kjellberg, Wadell et al. 2000; Nagai, Maehama
et al. 2000; Nobbenhuis, Meijer et al. 2001; Paraskevaidis, Koliopoulos et al. 2001;
Acladious, Sutton et al. 2002], whereas Hybrid Capture II (HC II) was used in 2 studies
[Kucera, Sliutz et al. 2001; Lin, Tseng et al. 2001]. HPV DNA detection was performed both
preoperatively and postoperatively in most studies, while postoperative only HPV DNA
detection was applied in two studies [Bollen, Tjong et al. 1999; Paraskevaidis, Koliopoulos et
al. 2001]. The duration of follow-up varied from 3-6 weeks postoperatively [Lin, Tseng et al.
2001]to 206 months for some individuals [Bollen, Tjong et al. 1999].
Different methods were also applied in order to confirm or exclude recurrent or residual
disease. In one study [Lin, Tseng et al. 2001], hysterectomy and pathologic examination was
performed 3-6 weeks after initial treatment. Cytology and/or colposcopy was applied in 5
studies [Elfgren, Bistoletti et al. 1996; Chua and Hjerpe 1997; Kjellberg, Wadell et al. 2000;
Kucera, Sliutz et al. 2001; Nobbenhuis, Meijer et al. 2001] (11,12,16,18,19), biopsy was
employed in 2 studies [Distefano, Picconi et al. 1998; Paraskevaidis, Koliopoulos et al. 2001]
and combination of these methods was employed in 2 studies [Bollen, Tjong et al. 1999;
Nagai, Maehama et al. 2000].
There were available data for 876 women who underwent conservative surgical treatment for
CIN.
HPV DNA was detected preoperatively in 718 of the 873 women (82.2%). The sensitivity of
the preoperative HPV test is particularly high in studies that included only high-grade lesions
[Chua and Hjerpe 1997; Nagai, Maehama et al. 2000; Lin, Tseng et al. 2001], where HPV
DNA is detected in 176 out of 181 cases (97.2%).
Among the 672 women in whom the treatment was considered to be successful, 566 (84.2%)
had a negative postoperative HPV DNA test, whereas 106 (15.8%) had a positive
postoperative HPV DNA test. In contrast, among the 204 cases that were considered as
treatment failures, only 35 cases (17.1%) had a negative postoperative HPV DNA test,
whereas 169 cases (82.8%) were positive for HPV DNA postoperatively. The sensitivity of
HPV DNA testing in detecting treatment failures was quite good in most studies, reaching
100% in four of them [Elfgren, Bistoletti et al. 1996; Bollen, Tjong et al. 1999; Nagai,
Maehama et al. 2000; Lin, Tseng et al. 2001], whereas a modest performance, in the range of
50%, was demonstrated in two studies [Distefano, Picconi et al. 1998; Acladious, Sutton et al.
2002]. The specificity of the test differed across the studies, ranging from 44% [Bollen, Tjong
et al. 1999] to 95% [Elfgren, Bistoletti et al. 1996].
In summary, there is evidence that HPV testing post treatment can more quickly and
efficiently detect a treatment failure.
Timing of taking a post treatment HPV test.
The optimal timing for a post-treatment HPV test has been explored in several studies, notably
Elfgren et al, 2002. A substantial proportion of women show clearance already at 3 months
and clearance is also significant between 3 and 6 months. After 6 months, the clearance rate is
slower, however. An extensive evaluation of possible post-treatment testig options in the
Dutch screening program that previously used posttreatment cytology at 6, 12 and 24 months
post treatment found evidence to suggest that double testing with cytology and HPV at 6
months and 24 months would be more efficient.
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RECOMMENDATIONS
With regard to the fact that, in at least some countries, women who have been treated for CIN
still have an increased risk for invasive cervical cancer, there is a definite need for improved
follow-up regimens.
The use of post-treatment HPV testing should be explored in design of new follow-up
regimens after CIN treatment.
Evidence supports the use of a double cytology and HPV test at 6 months post treatment for
improved safety of posttreatment follow-up. While there is evidence to suggest that
subsequent follow-up of women negative for both HPV and in cytology needs to be less
intense, evidence can not distinguish which specific follow-up regimen that should be used.
Implementation with careful monitoring and/or randomisation is recommended.
Further research on long-term protection of HPV-negativity as well as of joint cytology and
HPV-negativity is warranted.
7.4. Impact of HPV vaccination on design of cervical screening programs
7.4.1. Immune response to HPV infection and immunization
Studies in animal model systems performed over 40 years ago showed that there was a
protective effect against papillomavirus infection that is mediated by immune serum reactions
against the viral particle. For HPV, neutralizing antibodies can be generated by immunizing
animals with intact, infectious virus particles. This approach cannot be utilized in humans
because of the oncogenes contained in the HPV genome.
Natural HPV infection of the genital tract gives rise to a modest but measurable serum
antibody response in most individuals. There are a series of methodological issues that make
it difficult to unambiguously study whether immunity against type-specific reinfection occurs,
but significant (but not complete) protection against reinfection has been found to be
associated with the presence of HPV antibodies. A cell-mediated immune response also
occurs, as mentioned below.
A critical discovery in the development of vaccines to prevent HPV infection was that the L1
protein could be expressed in eukaryotic cells and could self assemble into so-called virus-like
particles (VLPs) [Zhou, Sun et al. 1991]. HPV L1 VLPs contain the same conformationally
dependent neutralizing epitopes as are present on infectious viruses. The L2 protein can also
be expressed with L1 protein in yeast or Sf9 cells, giving rise to “L1 plus L2” VLPs. So far all
available evidence indicates that both virus particles, L1 VLPs and L1 plus L2 VLPs react in
an undistinguishable fashion with immune sera, whether from natural infection or from
experimental infection in animals. VLPs can be purified and used to immunize animals and
humans by intramuscular injection. DNA plasmids or recombinant viruses (non-HPV)
designed to express L1 and other HPV proteins can also be administered by intramuscular
injection.
The immunogenicity of HPV particles involves presentation to the immune system of
conformational neutralizing epitopes displayed on viral capsids. Although the HPV types are
defined on the DNA sequence level, there is a very good correlation with serotypes, i.e.
individual HPV types have antigenically distinct epitopes and there is very limited crossneutralization.
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Vaccination of animals and humans with VLPs gives rise to neutralizing antibodies in serum.
In addition, antibodies to VLPs can be detected in cervical secretions of intramuscularly
immunized monkeys. However, the titer was less than 1% of the serum titer.
Young women who received an HPV 11 L1 VLP vaccine by intramuscular vaccination
developed serum antibodies that effectively neutralized infectious HPV 11.Within individual
HPV types, such as HPV 16, there are sequence variants, and there has been concern that
vaccination with VLPs may not afford protection against infection with various virus variants.
The immune response in human serum after natural infection is type-specific, but can not
distinguish between different variants of virus within the same type. Certain neutralizing
monoclonal antibodies are very effective for inhibiting the reactivity of immune sera from
humans against VLPs. The monoclonal antibodies that are able to block the natural antibody
response will react with particles from all different variants within a virus type, but not with
particles from other virus types. Also serum from human volunteers vaccinated with VLPs
will react with different virus variants. In a study of volunteers vaccinated with HPV 16 L1
VLPs effective neutralization a series of HPV16 variants was found. From a vaccine
perspective, HPV16 can therefore be said to constitute a single serotype.
HPV is transmitted by contact with desquamated keratinocytes from an infected individual.
This contact may be indirect, as in the case of cutaneous wart viruses, or sexual in the case of
cervical infection. These desquamated keratinocytes contain virus particles that escape the
cell and infect the new host. As there is no viremic phase, it is likely that the neutralizing
antibodies need to be present not only in serum but also in the immediate environment, such
as the cervix or external genital epithelium in order to prevent infection. Serum IgG
antibodies can reach the cervical epithelium and secretions by transudation. It is commonly
believed that exposure of the basal epithelial cell to infectious HPV results from microtrauma,
and the same process could damage to small blood vessels, increasing the antibody presence
in the cervical environment.
As indicated above, a cell mediated immune (CMI) response clearly occurs in natural HPV
infection in addition to an antibody response. This subject was extensively reviewed recently
by Man. Also, VLP vaccination of young women induces L1-specific T cell responses
detectable by proliferation of CD4+ and CD8+ T cells and in vitro production of Th1 and Th2
cytokines [Pinto, Edwards et al. 2003].
7.4.2. Vaccine trials in humans
Several vaccine trials are underway in humans, although there have been only a few published
results to date. Two large pharmaceutical companies (Merck, Sharp and Dome, and
GlaxoSmithKline in collaboration with MedImmune) are currently conducting vaccine trials
in the United States and elsewhere. In addition, the National Cancer Institute (United States)
is conducting trials in Costa Rica.
In a blinded, randomised, placebo-controlled dose escalation Phase I study of an HPV 11 L1
VLP vaccine, the vaccine was well tolerated and induced high levels of neutralizing
antibodies in serum.
In a Phase I vaccine trial using HPV 16 L1 VLPs, the vaccine was also well tolerated and all
subjects who received the vaccine seroconverted. Titers were approximately 40-fold higher
than what is observed in natural infection.
Recently, an interim analysis of a Phase II trial of an HPV 16 L1 VLP vaccine was reported
[Koutsky, Ault et al. 2002]. In this study, 2,392 women (16 to 23 years old) were randomized
to receive placebo or three doses of 40 g of HPV 16 L1 VLP vaccine produced in yeast cells
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at 0, 2 and 6 months. Genital samples for HPV DNA were obtained at enrollment and every
six months. Biopsy tissue was evaluated for cervical intraepithelial neoplasia and analysed by
PCR for HPV 16 DNA. The mean duration of follow-up was 17 months. The incidence of
persistent HPV 16 infection was 3.8/100 subject-years at risk in the placebo group and 0.0/100
subject-years at risk in the vaccine group (100 percent efficacy, 95 percent confidence
interval, 90 percent to 100 percent; p<0.0001). All nine cases of HPV 16-related CIN
occurred among placebo recipients. It was concluded that administration of the HPV 16
vaccine reduced the incidence of HPV 16 infections and HPV 16-related CIN. It should be
noted that the follow up of the study subjects continues, and it is likely that additional data on
the long-term immunogenicity and protective effects of the vaccine will be forthcoming.
7.4.3. Unresolved questions about HPV vaccine trials
Endpoints
It is a challenge to find appropriate endpoints for trials of HPV vaccines. Evidence of incident
HPV infection with a vaccine type is an endpoint that would seem to be an obvious choice for
a vaccine trial against an infectious disease. However, a high percentage of sexually active
women, especially those with more than four or five lifetime sexual partners are at least
transiently infected with one or more genital HPV types. Because HPV-induced clinical
disease occurs in only a relatively small proportion of infected individuals, estimates of
efficacy based on protection against infection can not be extrapolated to protection also
against clinical disease. Genital warts, or high-grade cervical dysplastic lesions of the cervix,
for example, probably occur in no more than 5% of infected individuals within a two to three
year period after infection. In addition, the clinical disease must be shown to be attributable to
the specific HPV present in the vaccine before conclusions can be drawn about success or
failure of protection. For example, numerous HPV types can cause cervical dysplasia.
Cervical dysplasia in a vaccine recipient may therefore be due to infection with a vaccine
HPV type, indicating lack of protection, or due to infection by a non-vaccine type. This is a
complex issue, and mandates the enrollment of large numbers of women in prospective trials
of vaccine efficacy.
Inclusion of different HPV types
Antibody responses elicited by VLP immunization are quite specific for the individual HPV
type, with limited cross-neutralisation even for closely related HPV types. At least 15
different “high risk” types have been identified as causative agents of cervical cancer. With
these considerations, an important question is “How many different HPV types can be
included, given that each type requires a certain amount of antigen to be included in the
preparation?”
Hughes et al, attempted to determine the theoretical impact of a prophylactic HPV vaccine
using mathematical models: one model focusing on HPV infection, and one model focusing
on cervical cancer. The authors determined that characteristics of sexual behavior and
characteristics of the vaccine affect the steady state prevalence of HPV that would be expected
if a vaccine program were begun. They determined that vaccinating women alone could
reduce the prevalence of infection with the specific HPV type in the vaccine by 30%
reduction, and that vaccinating both males and females could reduce the prevalence by 44%.
The analysis supported the concern that a “fill-in” effect is possible, that is, if a vaccine gave
protection against some- but not all - oncogenic HPV types, another oncogenic HPV type
might increase and may replace the disease caused by the types eliminated by the vaccine.
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They concluded that a multivalent vaccine containing the majority of disease-causing HPV
types would reduce the need for colposcopy and treatment, but that screening programs would
remain necessary unless the vaccines were very effective and were given to a large percentage
of the population. In contrast to less common sexually transmitted infections that are
restricted to defined core groups, an HPV vaccine would need to be given to a large
percentage of the population since HPV infection is extremely common.
To reduce cervical cancer by 90%, probably eight to ten types need to be included, assuming
little or no cross-protection by vaccination. However, it cannot be assumed that each HPV
type represented in a multivalent product would each be 100% effective. Also, numerous
other types that perhaps do not often cause invasive cancer are causative agents of cervical
dysplasia, and therefore cytological screening programs (and possibly HPV typing) will
continue to be necessary.
Vaccination against non-oncogenic HPV
Since the biology and means of prevention by VLP vaccination is presumably the same for the
benign HPV types as for the oncogenic ones, it is perfectly feasible to also vaccinate against
these viruses. HPV types 6 and 11 jointly cause more than 90% of genital condyloma
acuminata lesions, a common clinical condition of significant impact to sexual health. Low
grade dysplastic lesions of the cervix may be caused by these and other non-oncogenic types
as well. An additional reason for vaccinating against HPV types 6 and 11 is the fact that these
infections can cause serious disease in rare circumstances. HPV 11 is the major cause of
recurrent respiratory papillomatosis, a severe disease that may be fatal. So-called giant
condylomas or Buschke-Löwenstein tumors are usually caused by HPV 6. These tumors are
regarded as having a low potential for malignancy, but may also be fatal.
Several current vaccines undergoing testing include HPV 6, HPV 11, or both of these low risk
types. To date, although several vaccine trials are designed to test protection against low risk
types, no efficacy data is available.
Inclusion of HPV proteins in addition to L1 in vaccines
Addition of other proteins to the L1 VLPs requires increased technologic challenges and costs.
However, the L2 minor capsid protein is worthy of consideration, because antibodies elicited
to non-denatured L2 protein appear to have a degree of cross reactivity to heterologous HPV
types, and therefore may be able to protect the immunized individual against multiple HPV
types. The addition of early genes (E7 in particular) is also being investigated, to determine if
a cell-mediated immune response could be elicited along with the antibody response to the L1
VLP component [Greenstone, Nieland et al. 1998] [Nieland, Da Silva et al. 1999]. If so, this
would open the way to development of vaccines with a therapeutic component included, for
combined used in treatment and prophylaxis.
Duration and consistency of the antibody response to VLPs
The duration of protection against HPV infection has not been evaluated. Encouraging data
presented on the antibody response to HPV L1 VLP vaccines suggest only a slight decline in
the high serum antibody titers over a six to eight month evaluation period. The recombinant
vaccines that have been produced to prevent hepatits B infection may provide some insight
into the possible duration of protection against infection. For the hepatitis B vaccines,
neutralizing antibodies are induced in high titers that slowly decrease over a time span of
several years. Protection from hepatitis B infection appears to be long lasting (at least five to
ten years, perhaps much longer).
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Optimal timing for vaccination
Assuming the vaccines for HPV are eventually shown to be safe and effective, what would be
the optimal age for vaccination? Epidemiological studies indicate that many women become
infected within several months of initiation of sexual activity. Vaccination at an age of eight
to ten years may be appropriate, perhaps adding a booster in adolescence or early adulthood,
may seem like an obvious strategy. However, until the duration of the length of protection is
known it can not be properly modelled which targeting of specific age groups that would most
rapidly stop the spreading of the infection in the population.
Immunization of males
Genital tract HPV is sexually transmitted. Therefore, immunization of men may help prevent
infection in women. Currently there is little data available regarding the immune response to
HPV VLPs in men, although studies are being initiated. It is expected that immunization of
men with VLPs will elicit a serum immune response similar to that in women. Whether men
will be protected against infection or clinical disease resulting from infection (genital warts,
for example) has not been tested. A major obstacle in testing the efficacy of HPV vaccines in
men has been the lack of safe, inexpensive, and sensitive testing methods for HPV.
Antagonism between types
Although it is clear that multiple HPV types can be detected in the same individual, it is not
known if different HPV types can interfere with infection or pathogenesis by each other. The
possibility that different HPV types interfere with each other has been the subject of several
studies. The results suggest that infection with HPV 6 may interfere with HPV 16-associated
cervical carcinogenesis. Further studies are needed to clarify this potentially important issue
as new vaccine formulations are considered that contain numerous HPV types.
Conclusions and recommendations on HPV vaccines
Currently, only prophylactic HPV vaccines have shown promise. While prophylactic
vaccination is likely to provide important future health gains, cervical screening will need to
be continued for a whole generation of women that is already infected.
Due to the multiplicity of HPV types and the fact that the coming vaccines are type-specific,
the prophylactic vaccines are not likely to eradicate cervical cancer. A reduction in
background risk by elimination of the most important HPV types would affect costeffectiveness and timing/intervals of screening programs, but would not obviate them.
Continuous monitoring of which HPV types that are spreading in the populations will become
necessary, for early monitoring of “fill in” phenomena, inappropriate vaccination strategies or
other reasons for vaccination failure.
Research needed:
a) Monitoring of spread of HPV types in populations to be targeted by type-specific
vaccination.
b) Research on duration of efficacy of the vaccine.
c) Research on optimal vaccination strategies, preferentially using mathematical modelling
and/or implementation evaluation.
d) Research on optimal screening policies in vaccinated populations, particularly using
mathematical modelling.
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123
8. Glossarium and list of abbreviations
For the editor: test and programme accuracy measures are not defined here, see chapter 2.
Maybe all glossaria can be fused to one glossarium at the end of the guideline. As alternative
I propose to repeat the definitions that concern chapter 3 once again. (test- & programme
sensitivity, specificity, predictive values, sojourn time distribution, lead time).
AGUS: atypical glandular cell of unspecified significance
AIS: adenocarcinoma in situ
ASC-US: atypical squamous cells of unspecified significance
ASC-H: atypical squamous cells, high-grade squamous lesion cannot be excluded
CIN: cervical intra-epithelial neoplasia
CIS: carcinoma in situ
CP: conventional Pap smear
FDA: Food and Drugs Administration (United States)
FN: false negative
FP: false positive
HSIL: high-grade squamous intra-epithelial lesion
LBC: liquid based cytology
LSIL: low-grade squamous intra-epithelial lesion
NHS: National Health Services (United Kingdom)
PPV: positive predictive value
RCT: randomised clinical trial
SBLB: satisfactory but limited by
TBS: The Bethesda System
TN: true negative
TP: true positive
Verification bias: bias in the estimation of the diagnostic validity of a test when differential
verification (unequal fractions) of screen positives and negatives occurs
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10. Appendices
10.1. Appendix 1: Collection of adequate Pap smears (Marc ARBYN, Joe
JORDAN, Euphemia MCGOOGAN, Julieta PATNICK)
10.2. Appendix 2: Reporting schemes for cervical cytology (Ulrich SCHENCK,
Marc ARBYN)
A) National reporting schemes
B) Conversion between national and TBS cytological classification systems
C) Discussion on terminology
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