Key words
cervical cancer - cervical interepithelial neoplasia (CIN) - human papillomavirus
Schlüsselwörter
Zervixkarzinom - Zervixpräkanzerose - humane Papillomviren
Introduction
The role of HPV detection in cervical cancer screening is still discussed controversially
in Germany. One of the two main topics of last yearʼs EUROGIN (European Research Organisation
on Genital Infection and Neoplasia) conference, held in Lisbon in May 2011, was –
in addition to HPV vaccination – the importance of HPV detection. The four-day conference
focused less on the question whether HPV testing should be carried out as part of cancer screening. Many of the papers
focused instead on the possibility of a more differentiated assessment of HPV infection
based on the confirmation of individual high-risk HPV genotypes (hrHPV).
Procedures for HPV Testing and Genotyping
Procedures for HPV Testing and Genotyping
After its approval by the US Food and Drug Administration (FDA) in 2003, the Hybrid
Capture 2 test (hc2; Qiagen, Hilden, Germany) has been the standard test for the detection
of HPV in clinical practice for almost 10 years. The test can detect 13 hrHPV genotypes
(16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) and is based on the hybridisation
of HPV DNA with HPV RNA. The DNA-RNA connections are marked with antibodies and detected
using chemiluminescence. However, the test does not differentiate between individual
HPV genotypes. Results are given as either hrHPV-positive or hrHPV-negative. The assay
can also be used to detect low-risk HPV (lrHPV) genotypes. The advantage of the hc2
test is its long-standing and extensive use in clinical practice; the disadvantage
is the potential for cross-reactivity with other, usually lrHPV genotypes, which can
lead to false-positive results [1].
In the last decade numerous new hrHPV tests have come on the market based on DNA detection
using polymerase chain reaction (PCR) assays. These tests are usually capable of detecting
any of 14 hrHPV genotypes (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Moreover,
some offer the possibility of separately determining the individual hrHPV genotype.
In general, the most commonly detected genotypes are HPV 16 and 18, and some manufacturers
also offer tests which can detect other genotypes. In principle, it is possible to
detect even very low amounts of virus DNA using PCR. Such highly sensitive detection
methods, however, have no importance in routine clinical diagnostics as they often
detect HPV infections with no clinical relevance (cf. [Table 1]). Most commonly available, commercial PCR-based tests have therefore adapted their
detection thresholds accordingly. The disadvantage of PCR-based assays is the limited
clinical experience,
although numerous studies of these tests have been done in recent years [1], [2].
Table 1 Definitions.
|
Sensitivity
|
Number of persons with disease (in this case, women with high-grade CIN), who were
correctly identified by the test method used (HPV test or cytological smear) as having
disease compared to the overall number of persons with disease.
|
|
Specificity
|
Number of healthy persons who were correctly identified by the test method as free
from disease compared to the overall number of healthy persons in the study.
|
|
A test with maximum sensitivity would detect all persons with disease: the test sensitivity
would be 100 %. However, such a test usually has a low specificity, i.e. many false-positive
results.
|
|
Positive predictive value (PPV):
|
Number of persons with de facto disease out of all persons with positive test results.
In younger women, HPV test has a relatively low PPV for high-grade CIN, as many transient
HPV infections may occur in this age group without dysplastic changes of the cervix.
|
|
Negative predictive value (NPV):
|
Number of de facto healthy persons of all persons with negative test results. The
NPV of a correctly performed HPV test is very high as almost no cervical cancers or
high-grade CIN lesions are HPV-negative; if the smear is HPV-negative, any such changes
can be excluded with a high degree of probability.
|
|
Cytology triage after positive HPV test:
|
All women first tested for HPV. HPV-positive women additionally investigated by conventional
Pap smear or liquid-based cytology. Women with abnormal cytology are investigated
further, usually using colposcopy, while women with normal cytology are investigated
again by HPV test and poss. cytology after a specified interval of time has passed.
|
HPV-based Screening Compared to Cytology
HPV-based Screening Compared to Cytology
Several recent large European randomised controlled studies were able to demonstrate
the superiority of HPV-based cervical cancer screening compared to cytology – whether
using HPV detection alone or in combination with cytology (cf. [Table 2]). However, it is important to consider the results with regard to their specificity
and positive predictive value (PPV) as these are important parameters for feasible
screening programmes.
Table 2 Overview of randomised controlled studies on cervical cancer screening by HPV detection.
|
Study
|
Country
|
No. of partici-pants
|
Age
(years)
|
Screening method used
|
Sequence
|
Colposcopy rate
|
Follow-up (years)
|
Results and comments
|
|
HPV group
|
|
|
hc2: Hybrid Capture 2 HPV test; PCR: HPV detection with PCR assay; LBC: liquid-based
cytology; Pap: conventional cytology; vs.: versus; n. s.: not specified; ICC: invasive
cervical cancer; AIS: adenocarcinoma in situ
|
|
ARTISTIC [15]
|
UK
|
24 510
|
20–64
|
hc2 + LBC
|
LBC
|
2 investigations with an interval of 2 years
|
n. s.
|
4.8
|
1st round: no differences
2nd round: signif. fewer CIN 2+/CIN 3+ in HPV group, but small absolute difference
|
|
Finland [16]
|
Finland
|
58 076
|
30–60
|
hc2 + Pap triage
|
Pap
|
1 investigation, follow-up via cancer registry
|
hc2: 7.2 %
Pap: 6.6 %
|
3.3
|
1st round: ⅓ more CIN 2+ and CIN 3+ in HPV group
After 3.3 Y: HPV: 11 ICC/AIS vs. Pap: 6 ICC/AIS
|
|
NTCC phase 1 [5]
|
Italy
|
45 174
|
25–60
|
hc2 + LBC
|
Pap
|
2 investigations with an interval of 3 years
|
hc2: 9.4 %
Pap: 3.0 %
|
3.5
|
1st round: 2× more CIN 2+ and CIN 3+ in HPV group; similar number of ICC
2nd round: HPV: 0 ICC vs. Pap: 9 ICC (p = 0.004)
Age 35–60 yrs: 1st round HPV: twice the number of CIN 2 & 3; 2nd round HPV: ca. half
the number of CIN 2 & 3
|
|
NTCC phase 2 [5]
|
Italy
|
49 196
|
25–60
|
hc2
|
Pap
|
2 investigations with an interval of 3 years
|
|
|
|
|
POBASCAM [4], [17], [18]
|
Nether-lands
|
44 105 (1st round)
33 499 (2nd round)
|
29–56
|
PCR + Pap
|
Pap
|
2 investigations with an interval of 5 years
|
PCR: 2.3 %, 1.3 %
Pap: 1.3 %, 1.9 %
|
6.5
|
1st round: 24 % more CIN 2+ in HPV group
2nd round: 28 % fewer CIN 3+ and 71 % fewer ICC in HPV group
→ with PCR earlier detection of clinically relevant CIN 2+
|
|
Swedescreen [3]
|
Sweden
|
12 527
|
32–38
|
PCR + Pap
|
Pap
|
2 investigations with an interval of 3 years
|
PCR: 1.6 %
|
4.1
|
1st round: 51 % more CIN 2+ in HPV group
2nd round: 42 % fewer CIN 2+ in HPV group
|
|
India [19] (underdeveloped region, no previous screening programme)
|
131 746
|
30–59
|
single-round of screening:
hc2 vs. Pap vs. cervical inspection with acetic acid vs. “standard care”
|
hc2: 1.3 %
Pap: 4.9 %
insp.: 10.8 %
|
8
|
relative risk for the detection of advanced cervical cancer (≥ II) and the number
of deaths from cancer halved with HPV vs. control group (not with Pap or cervical
inspection)
|
Between 1997 and 2005, the extensive Swedish population-based research programme Swedescreen
evaluated 11 different screening algorithms based either on HPV tests alone, or on
Pap tests alone, or on various combinations of HPV and Pap. In a subgroup of women
aged 32–38 years from the Swedescreen collective, a 35 % increased sensitivity for
the detection of CIN 3 and invasive carcinoma (CIN 3+) was demonstrated in the group
which underwent “Pap and type-specific HPV persistence” double testing compared to
cytology alone [3]. The positive predictive value was also comparable. But this screening strategy
is expensive and time-consuming as twice as many tests are required to obtain these
good screening results. Another algorithm used the HPV test as the initial test, followed
by a cytology triage in HPV-positive women with subsequent testing for persistent
HPV infections in Pap-negative women and compared this algorithm with the results
of cytology
alone. This algorithm achieved an increase in sensitivity of 30 % for the detection
of CIN 3 or invasive carcinoma (CIN 3+). Moreover, this differential diagnostic algorithm
had a comparable positive predictive value (PPV) but the number of screening tests
required only increased by 12 % (from 6257 to 7019).
The Dutch POBASCAM study also demonstrated a higher sensitivity for combined screening
compared to cytology alone [4]. Two screening phases were carried out with an interval of 5 years between phases.
In the 1st phase 24 % more CIN 2+ were detected in the group who underwent combined
cytology and HPV tests compared to the group tested using cytology alone. In the 2nd
phase, the CIN 3+ detection rate in the HPV-tested group was 28 % lower and the rate
of invasive cancers was 71 % lower compared to the cytology group despite comparable
overall numbers of CIN 3+ in both groups. These data show that combined screening
can result in earlier detection of clinically relevant intraepithelial neoplasias.
The Italian NTCC study showed similar results [5]. A total of 47 000 women were randomised into two groups, with the 1st group screened
by cytology alone and the 2nd group screened using cytology and HPV test (later in
the study, no cytology was done in later recruits to the 2nd cohort). The detection
of abnormalities was followed by the appropriate therapy. After 2 years, both groups
again had cytological screening. In the 1st screening, a similar number of invasive
cervical carcinomas had been detected in both groups ([Fig. 1]). In the 2nd screening, no further cancers were found in the group screened using
HPV test, while 9 more cancers were detected in the cytology group. The initial cytology
findings in all 9 cases with carcinomas found at the 2nd screening had been normal.
Fig. 1 Squamous cell carcinoma of the uterine cervix pT1b1, N0, G2 after radical endoscopic
hysterectomy and combined blue dye/technetium 99m radiocolloid (intracervical) identification
of sentinel lymph nodes.
With regard to CIN 2 and CIN 3 ([Fig. 2]), almost twice as many cases were found in the HPV group in the 1st round of screening
and approximately half as many in the 2nd round compared to the cytology group. This
led the authors to conclude that initial HPV screening results in better early detection
of clinically relevant precancerous lesions. The detection of twice as many CIN 2
and CIN 3 with HPV test during the initial screening round meant that these lesions
were treated in time and progression to more invasive cancers was prevented. It must
be assumed that the precancerous lesions not detected during the 1st screening phase
in the cytology group led to the 9 cancers found at the 2nd screening. However, the
authors recommend that screening including HPV testing should only be done in women
older than 35 years, followed by cytology triage in HPV-positive cases.
Fig. 2 Colposcopy of a CIN 2 lesion after application of 5 % acetic acid. Insert shows diagnostic
fluorescence imagíng of the same CIN 2 after the application of hexaminolevulinate.
In 2008, Dillner et al. investigated the 6-year risk to develop a CIN 3+ lesion as
a function of hrHPV status and cytology in a multinational cohort study [6]. No HPV genotyping was performed in this study. As expected, hrHPV-positive women
with abnormal cytology had the highest risk with 34 %. In hrHPV-positive women with
normal cytology the CIN 3+ rate after 6 years was 10 %, while in the converse case
(i.e., hrHPV negative, abnormal cytology) the incidence was only 2.7 %. Women with
a negative hrHPV status at the beginning of the study showed a 6-year risk to develop
CIN 3+ of only 0.27 % – irrespective of their initial cytological findings. The authors
therefore concluded that if the initial hrHPV result was negative, the screening interval
could be extended to 6 years without additional risk for the women.
Risk Potential for Different hrHPV Types
Risk Potential for Different hrHPV Types
Numerous retrospective and a few prospective studies have shown clear differences
between various hrHPV genotypes with regard to the risk of developing CIN or invasive
cancer. In one of the largest retrospective studies of women with invasive cervical
cancer performed worldwide, de Sanjose et al. were able to determine the HPV DNA in
85 % of the more than 10 000 investigated cases [7]. HPV 16 was by far the most commonly found genotype with an incidence of 61 %, followed
by HPV 18 (10 %) and HPV 45 (6 %). Moreover, the women in whom one of these 3 HPV
genotypes was detected were approximately 4 years younger when the cancers were detected
compared to women in whom other HPV genotypes were determined in their cancer cells.
An American study of 1213 women with carcinoma in situ (CIN 3) and 808 women with
invasive cervical cancer detected HPV DNA in 97.1 % of CIN 3 lesions and in 91.0 %
of invasive carcinomas [8]. Once again, HPV 16 was the most common genotype with an incidence of 56.3 and 53.2 %,
respectively. In cases with CIN 3 this was followed by HPV 31 (12.6 %), HPV 33 (8.0 %)
and HPV 18 (5.9 %), and HPV 18 (13.1 %) and HPV 45 (6.1 %) were also detected in women
with invasive cancer. This study also found a significant difference in age between
women with invasive cancer and positive for HPV 16 or HPV 18 compared to women with
other HPV genotypes (48.1 and 45.9 years respectively vs. 52.3 years).
HPV 16 also played the most important role in direct precancerous lesions. In the
American ALTS study, HPV DNA was detected in 98.8 % of the 608 investigated women
with CIN 3 [9]. 59.9 % of cases were HPV 16 positive. In this study, HPV 18 (13.2 %) was only the
fifth most common genotype after HPV 31 (18.1 %), HPV 52 (14.8 %) and HPV 51 (14.0 %).
Here again, precancerous lesions caused by HPV 16 occurred in younger women (23.5
years) compared to lesions caused by other genotypes (25 years). Interestingly, HPV
16 positive CIN 3 lesions were more common in women who smoked and less common in
multiparous women.
In 2005 Khan et al. showed in a prospective type-specific HPV cohort study of more
than 20 000 women without high-grade cytology that HPV 16 and HPV 18 positive women
had a significantly increased risk of developing CIN 3+ [10]. For women who were HPV 16 positive at the beginning of the study the cumulative
10-year incidence rate for CIN 3+ was 17 %, and for HPV 18 positive women it was 14 %.
In comparison, the risk for other hrHPV genotypes was 3 %, and for hrHPV-negative
women it was only 1 %. This risk distribution was even more significant for women
above the age of 30. In HPV 16 positive women aged > 30 years the 10-year incidence
rate for CIN 3+ was 20 %, while HPV 18 positive women aged > 30 years had a 10-year
incidence rate of 15 %.
The higher sensitivity of HPV tests for screening compared to conventional cytology
was also clearly demonstrated. The authors compared women above the age of 30 with
abnormal cytology irrespective of their HPV status (LSIL: low grade squamous intraepithelial
lesions; women with high-grade cytology were not included in the study) with women
who were HPV 16 positive or HPV 18 positive at the beginning of the study. The 10-year
incidence for CIN 3+ in the abnormal cytology group was only 11 % compared to 20 %
and 15 % respectively in the HPV 16 and HPV 18 groups. Moreover, even women with normal
cytology who were HPV 16 or HPV 18 positive were shown to have an increased risk for
developing CIN 3+.
Based on these data the authors advocate that type-specific HPV 16 and HPV 18 testing
be done when screening women above the age of 20, and recommend colposcopy if the
result is positive. If the findings are positive for other hrHPV genotypes they consider
re-screening after 1 year sufficient.
Another recently published follow-up investigation of the same study collective showed
a very low cumulative probability of developing CIN 3+ in women with a negative HPV
status [11]. The incidence for women under the age of 30 was 1.8 %, while for women above the
age of 30 it was merely 0.7 % over a follow-up period of more than 15 years. The increased
risk for HPV 16 positive women to develop CIN 3+ was again confirmed in this study.
Compared to other hrHPV genotypes, the risk in women aged more than 30 years was more
than double (2.7-fold relative risk), and compared to HPV negativity it was more than
6 times as high (6.2-fold relative risk). To compare the independent validity of HPV
testing with that of cytology, the authors calculated the respective relative risk
of developing CIN 2+ after a positive compared to a negative hrHPV test (7.1 for age
< 30 years and 8.5 for 30+) and with abnormal (ASC-US/LSIL) compared to normal cytology
(NILM) (1.5 for women aged < 30 years and 2.9 for women aged 30+). This clearly shows
the higher validity of positive HPV test results compared to abnormal cytology results.
Because of the very high negative predictive value (NPV) of a negative HPV test in
this investigation, the authors concluded that for women above the age of 30 with
a negative HPV test and normal cytology, re-examination after 3 years – as is currently
the standard in the USA – may not be necessary that early.
A population-based prospective Danish cohort study started in 1991 came to similar
conclusions [12]. Around 7500 women with normal cytology at the start of the study underwent 2 gynaecological
investigations with Pap smears and hrHPV genotyping at an interval of 2 years between
investigations. The start of the study was defined as the time of the second investigation.
All citizens in Denmark have a personal identification number, and the country additionally
has a national pathology database in which all cervical cytologies and histologies
are entered. This allows such study collectives to be followed up with almost no dropouts
and at a relatively low cost. The evaluation of study results was done in 2007 after
a maximum follow-up of 13.4 years.
It was shown that one quarter (26 %) of the women who were HPV 16 positive at the
2nd investigation developed a CIN 3+ lesion during the follow-up period. For HPV 18
the incidence was 15.4 %. The figures were much lower for other hrHPV genotypes (HPV
33: 12.8 %; HPV 31: 9.8 %; HPV 35: 9.1 %; HPV 58: 8.3 %; HPV 45: 6.4 %). For hrHPV-negative
women the CIN 3+ risk after 12 years was 3.0 %.
Taking 2 cervical smears at an interval of 2 years allowed the authors to evaluate
the impact of persistent HPV infection. HPV 16, 31, 33, 35, 52 and 58 had the highest
prevalence at the beginning of the study, a higher rate of persistence and a higher
potential, if they persisted, to lead to a CIN 3+ lesion. Thus, 29.4 % of HPV 16 infections
were persistent, i.e. HPV 16 was detectable at both investigations, and 46 % of women
with persistent HPV 16 infection developed CIN 3+. Interestingly, the 12-year risk
for CIN 3+ in women in whom hrHPV was detected at both investigations using hc2 was
19.3 % irrespective of the HPV genotype, and thus similar to the risk reported for
women in whom HPV 16 was detected once (17.3 %).
In 2011 in a subgroup analysis of the ATHENA HPV study, in women with ASCUS cytology
(atypical squamous cells of undetermined significance) Stoler et al. found a clear
correlation between their HPV 16 and/or HPV 18 positivity rate and a more severe grade
of CIN [13]. The two HPV genotypes were found in only 8 % of women without CIN, but in 18 %
of cases with CIN 1, 44 % of cases with CIN 2 and 61 % of cases with CIN 3+. The absolute
risk for CIN 3+ was 20 % for HPV 16 positive women. Compared to HPV-negative women,
the relative risk for CIN 3+ was 70.9 for HPV 16 positive women and 15.4 for HPV 18
positive women. The relative risk for 12 other hrHPV genotypes was only 15.7 in total.
When HPV 16 was compared with these 12 other hrHPV genotypes, HPV 16 still had a relative
risk of 4.5.
Discussion
The studies presented here clearly show the potential for HPV tests in cervical cancer
screening. On the one hand, a negative result, particularly in women above the age
of 30, offers a high degree of certainty that they are unlikely to develop CIN 3+
within a period of several years. For these women, extending the intervals between
screenings, as recommended by some authors, is certainly justified. In view of the
pressure to reduce costs in healthcare today, this is additionally relevant.
The appropriate management of women who tested positive for hrHPV is more difficult.
A differentiated assessment on a case-by-case basis which also takes account of cytology
findings is necessary. HPV genotyping also offers advantages for the assessment of
the individual risk of an HPV-positive woman to develop CIN 3+ in the future. In all
of the studies cited here, HPV 16 was found to be the genotype with the highest oncogenic
potential among the hrHPV genotypes. It was not merely that in most cases HPV type
16 was detected in women with CIN 3 or carcinoma; prospectively, infection with HPV
16 resulted more often in the development of an intraepithelial neoplasia than other
hrHPV genotypes. The age at which HPV 16 was contracted was also lower than the age
at which women were infected with other hrHPV genotypes.
The studies did not come to any consistent conclusion with regard to other hrHPV genotypes,
doubtlessly also due to regional differences. In addition to HPV 18, the most important
genotype after HPV 16, HPV 31, 33 and 45 in particular appear to be associated with
a slightly higher risk. In addition, HPV genotyping also allows the definitive identification
of women with persistent infection, a significant risk factor for developing higher
grade lesions.
In clinical practice this could mean that women positive for HPV 16 and possibly also
women positive for HPV 18 could undergo colposcopy examination and be followed up
more closely than women with other hrHPV genotypes. Nevertheless, it is important
to keep in mind that the long-term risk of developing CIN 3+ is only 20–25 %, depending
on the study, after the first-time detection of HPV 16 [10], [12], [13]. For three quarters of HPV 16 positive women this entails unnecessary additional
investigations. However, if HPV 16 persists, the risk of developing a CIN 3+ lesion
increases to almost 50 % [12]. The more intensive management of women positive for HPV 16 places a burden on patients
and physicians and is obviously also accompanied by higher treatment costs. Whether
these additional costs can be compensated for by the reduced number of screenings
required by
HPV-negative women remains to be seen.
When interpreting the cited studies it is also important to remember that the results
obtained in some cases may not be easily transferred to the German early detection
system, as other countries already use distinctly longer screening intervals in the
setting of an organised screening programme. In the Dutch POBASCAM study, for example,
the interval between 2 investigations was 5 years, while in the Italian NTCC study
the interval was 2 years.
The data presented here may soon have practical consequences in the Netherlands. In
May 2011, the Health Council of the Netherlands which advises the Dutch government
revised its recommendations on cervical cancer screening. The new recommendations
suggest carrying out initial HPV-based screening, with around 5 tests done over the
lifetime of a woman: the first one at the age of 30 years, then at ages 35, 40, 50
and 60 years. HPV-positive women will be investigated further using cytology triage
[14].
Summary
In summary, it is clear that the inclusion of HPV testing in cervical cancer screening
offers a high degree of certainty. For women who are HPV-negative, the intervals between
investigations can be extended. HPV genotyping is currently being discussed with a
view to a better risk stratification and identification of women at increased risk
who will require more intensive monitoring.
