May 2009 - SOGC

N o 18 May 2009
What clue led you to believe that HPV plays a
causal role in the genesis of cervical cancer?
In 1969/70 we detected and purified Epstein-Barr virus
(EBV) in Burkitt lymphoma and nasopharyngeal cancers,
which meant we had the technology in place to look
for viral DNA in other tumour systems. Cervical cancer
looked very promising because its epidemiology strongly
indicated a link to a sexually transmitted disease. Herpes
simplex virus (HSV) had been the chief suspect since
1967, but as we failed to detect HSV-DNA in cervical
cancer we started to look for other viruses. Following
reports about the malignant conversion of condylomata
acuminata, I speculated that papillomavirus could be
responsible for cervical cancer. When Lutz Gissmann
discovered, and Ethel-Michele de Villiers cloned, HPV-6
in condylomas, we learned that papillomaviruses are
very heterogeneous and were disappointed that we
could not find HPV-6 in cervical cancers. However,
HPV-11 could be identified easily with HPV-6,
and with HPV-11 we received the first positive
results –weakly hybridizing signals– in cancer.
Matthias Dürst and Michael Boshart, by
then students in my research group, were
asked to clone these bands. Both were
very talented and discovered HPV-16
and -18 DNA.
(continues on page 3)
Newsletter
on Human
Papillomavirus
HPV researcH: THe forward looking
Views of nobel Prize laureaTe Harald
zur Hausen
Monograph
HPV-DNA integration
interview with
Harald
zur Hausen
hPV in screening and triage
HPV testing in routine cervical
cancer screening
www.hpvtoday.com
CASE STUDY
Vulvar cancer in the
anterior fourchette in
a young woman

2
EDITORIAL
nobel Prize for Harald zur Hausen
The Nobel Prize for Harald zur Hausen had been expected for many
years but the announcement that it had finally become reality was
an exciting honour for the laureate himself, his thesis and the whole
field of papillomavirus research. Zur Hausen went against dogma
when he postulated that certain HPV caused cervical cancer, the
second most common cancer among women. He realized that
HPV-DNA could exist in a non-productive state in the tumours
and, with the support of his staff, found HPV to be a heterogeneous
family of viruses. His discovery led to characterization of the
natural history of HPV infection, an understanding of mechanisms
of HPV-induced carcinogenesis, the standardization of HPV-DNA
testing systems and the development of prophylactic vaccines
against HPV acquisition.
For a long time surprisingly few people outside the international
papillomavirus (PV)-science community seemed to be interested
in the exciting discoveries of how a virus causes cancer and how
this knowledge could be used for a better prevention of various
human diseases. Two main topics finally brought PV-research to the
attention of public interest. One was research and recognition on
the usefulness of HPV-testing in the prevention of cervical cancer.
More recently, HPV vaccines and proposals of mass vaccination
made HPV a topic of genuine public interest.Can we be satisfied
with the achievements since zur Hausen's thesis?
In Germany more than 800,000 girls and young women, or more
than one third of the target group, are already vaccinated. More
than 40 million doses of HPV vaccine were given worldwide in
the years 2006-2008. On the other hand, following reports of
coincidental death cases, many girls and young women did not
complete their three doses of vaccine. Similarly, HPV-testing is
widely used and recommended by German guidelines in the triaging
of atypical smears. However, the German reality for women
with atypical smears is to be followed by repeat Pap smears or
to be transferred directly for cold knife conization without any
histological assessment by colposcopy prior to invasive treatment.
The chance of receiving false atypical Pap smear results is very high
for women who attend for annual screening smears, but the one
year interval for Pap smears is still demanded by the German law.
A revision of the German concept of cervical cancer prevention is
obviously necessary.
Although we cannot be fully satisfied, the field is now encouragingly
active.
PV scientists all over the world have added to the understanding
of carcinogenesis in general, HPV etiology in organs other than the
cervix, have shown new ways of cancer prevention and have developed
new types of vaccines. Time will prove the correctness of the
predicted decline in HPV-related diseases in vaccinated individuals
and the cost efficiency of HPV testing for primary cervical cancer
screening. With cheaper and better vaccines ahead, and improved
screening and new therapies in the pipeline, tumours induced by
HPV may face extinction within a few decades. This at least seems
more likely today than the thesis that papillomavirus causes cervical
cancer seemed to many just a few decades ago.
Karl Ulrich Petry
Frauenklinik im Klinikum Wolfsburg, Germany.
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Harald zur Hausen
Deutsches Krebsfroschungszentrum, Heidelberg, Germany.
(from page 1)
More than 30 years lay between the publication of
your thesis and the winning of the Nobel prize. Did
you have the impression that your concept of the
genesis of cervical cancer, which has since been
confirmed many times, was always accepted and
its importance always understood?
The hypothesis was accepted by German research agencies
from the very beginning and we received grants as
early as 1972 in support of our research. But I could
sense a significant refusal to accept the papillomavirus
concept among scientists adhering to herpes simplex
type 2 as a potential candidate causing cervical cancer. I
remember international conferences on tumour virology
in 1974 and 1975 where I received very strong resistance
from some of the herpes simplex researchers.
Besides cervical cancer, subsets of cancers of the
vulva, vagina, anus, penis, skin and oropharynx are
associated with HPV. How important is the role of
HPV in these tumours?
Papillomavirus DNA persistence of high risk types was
early evidence for a role of these viruses in cancers of
the vagina, vulva, anus and penis. However, a number
of these tumours still test HPV negative, especially
cancers of the penis and vulva. This raises the question
as to what lies behind these tumours. I assume that the
percentage of HPV-positive tumours of the oropharynx,
which has reached 60% in some publications, is too high
and might be due to contamination from non-malignant
virus-producing lesions of the same patient. One should
analyse this again very carefully based on viral RNA
synthesis because this would be a reliable proof that
these viruses really play a role in the genesis of these
cancers. I assume that only 25-30% of oropharyngeal
cancers, especially cancers of the tonsils, are high risk
HPV (HR HPV) positive.
What do you expect from current and future HPV
vaccines?
I expect a great deal from the vaccines, especially a significant
decline in HPV-16 and -18 precancerous lesions in
vaccinated women. Approximately 140,000 cone biopsies
are performed in Germany every year, which is a huge
number. Vaccine opponents should be aware that the
reduction of precancer is an important issue for young
women and societies. For developing countries, I expect
a drop in vaccine prices. I have heard that companies
have started talks about special prices for Third World
countries. Maybe we all should shout even louder that
the vaccines are currently too expensive! I see the first
attempts in China to develop their own HPV vaccines,
and there are rumours about an octavalent HPV vaccine
under development in India. These are encouraging signs
that prices will eventually fall drastically.
You opened the eyes of scientists who explored the
cause of cervical cancer. Today, could you help us
again with a vision of what we should look for in
the next few years?
I would go beyond the field of papillomavirus research.
The new discovery of a polyomavirus in Merkel cell
tumours will gain great importance in the future. These
viruses seem to be widespread, and it will be very
interesting to analyze the spectrum of tumours with
which they are associated. I see future opportunities in
the exploration of haematopoetic diseases, especially
leukaemias and lymphomas, and I would even speculate
on a possible involvement of viruses in breast cancer.
There are a number of interesting findings that may point
to a possible role of endogenous retroviral activation in
the genesis of breast cancer.
... The condyloma agent has been entirely
neglected thus far in all epidemiological
and serological studies relating not only to
cervical and penile, but also to vulvar and
perianal, carcinomas. This is particularly
unusual in view of the localization of
genital warts, their mode of venereal
transmission, the number of reports on
malignant transition, and the presence of
an agent belonging to a well-characterized
group of oncogenic DNA viruses.
zur Hausen H. Cancer Res 1976;36:794
As consumption of red meat is a risk factor for the development
of specific gastrointestinal cancers, it needs to
be explored whether this risk is really exclusively linked
to the carcinogenic chemicals that develop during the
cooking or barbecue processes, or whether hitherto
unknown viruses may also play a role in this carcinogenesis.
The same carcinogens are also formed when
poultry meat is cooked or barbecued, but as far as I am
aware white meat consumption is not associated with an
increased risk for gastrointestinal tumours as has been
observed for red meat –this does not fit together.
What are your personal future plans?
I would like to stimulate young scientists to look into
possible links between human cancers and infectious
agents more carefully. Because of the intensive
investigation of inherited cancers and cancers with
genetic modifications, the fact that there can be no
virus-induced cancer without modifications of the host
genome is often overlooked. Such modifications must
happen to establish malignant growth. In other words,
specific chromosomal aberrations in breast cancers do
not exclude a hidden virus in the background that is
activated by these chromosomal changes.
3

4
The sentinel concept is
well established for treating
patients with melanoma and
breast cancer, although in
breast disease the negative
predictive value is only of
90%. We conducted a prospective
study which included
more than 500 patients with
cervical cancer with the
hypothesis that sensitivity
should be at least 90% and
the negative predictive value
at least 99%.
In our cohort, the overall sensitivity
was only 77% and the
negative predictive value 94%.
About half of the patients
had tumours with a diameter
of 2 centimeters or less. The
sensitivity in this subgroup
was of 90% and the negative
predictive value 99%, 1 which is a strong indication that
the sentinel concept may be valid for this subgroup.
Since the prevalence of lymph node metastasis in
patients with tumours of ≤2 centimeters is 10%, a
sensitivity of 90% and a negative predictive value of 99%
would leave 1 patient out of 100 with a false-negative
result. However, in 81 out of 100 patients only the sentinel
lymph nodes would be removed and these patients
would profit from the concept.
Lymph node metastases in patients with cervical cancer
are more difficult to diagnose and treat than in patients
THe senTinel concePT
in PaTienTs wiTH
cerVical cancer
Achim Schneider*
Matthias Dürst**
*Department of Gynaecology, Charité, Berlin, Germany.
** Department of Gynaecology, Friedrich-Schiller-
University Jena, Germany.
Figure 1
Blue dye staining of a sentinel lymph node and its supporting lymph vessel
at the bifurcation of the right iliac vessels.
with breast cancer. Thus,
false-negative results must
be avoided.
Sentinel lymph nodes can
be evaluated by serial sectioning
(ultrastaging) with or
without immunostaining. This
approach allows to detect
micrometastases (>2 millimeters
but ≤2 millimeters)
that may be of prognostic
relevance to be detected.
Alternatively, molecular markers
such as CK19 mRNA have
been shown to be useful for
the detection of metastases
in the axillary lymph nodes in
patients with breast cancer,
and may replace frozen section
in the near future.
However, we showed that
for pelvic lymph nodes CK19
mRNA is specific only in cases which show clear histologic
evidence for metastasis. In contrast, HPV mRNA of
the E6 oncogene is highly specific for micrometastases
and small tumour cell clusters. 2 Experimentally, the HPV
mRNA assay has a sensitivity of one tumour cell in a
background of 10 5 normal cells. On the basis of this
highly specific marker, we are currently evaluating the
prognostic significance of HPV mRNA in the sentinel
lymph nodes of over 400 patients whose systematic
lymphadenectomy was negative by conventional
histology.
Figure 2
Partially dissected sentinel lymph node in the obturator fossa
References: 1. Altgassen C et al. J Clin Oncol 2008;26(18):2943-51. 2. Häfner N et al. Int J Cancer 2007;120(9):1842-6.

Peter Hillemanns
Department Obstetrics and Gynecology.
Medizinische Hochschule Hannover
(University) Hannover, Germany.
wHaT is THe cosT-effecTiVeness of HPV
VaccinaTion in germany?
The development of a prophylactic vaccine against HPV
is a major breakthrough in the prevention of invasive
cervical cancer. The first prophylactic tetravalent HPV
recombinant vaccine (HPV types -6,-11,-16,-18) was
granted a marketing authorisation in the European
Union in 2006. In 2007 a bivalent vaccine (HPV types
-16 and -18), indicated for the prevention of cervical
intraepithelial neoplasia (CIN) grades 2 and 3 and cervical
cancer causally related to HPV types -16 and -18, was
approved for use by the European Medicines Agency.
Both vaccines have been shown to be highly effective
in large phase- III clinical trials. 1,2
There has been an extensive discussion about the
costs of both HPV vaccines in Germany. A recent study
addressed this issue and conducted a cost-effectiveness
analysis.
An empirically calibrated Markov cohort model of the
natural history of HPV was used to assess the costeffectiveness
of the vaccine administered to 12-year-old
girls alongside existing cervical screening programs
in Germany. 3 The model estimated lifetime risks of
cervical cancer, CIN and genital warts and total lifetime
healthcare costs, life years gained and quality adjusted
life years (QALY) gained. The analysis was conducted
from the perspective of the German healthcare payer.
One-way sensitivity analysis was performed to explore
the impact on cost-effectiveness of varying a range of
key parameters for duration of vaccine protection, vaccine
and administration costs, Pap sensitivity, utilities,
discount rates and screening coverage. The impact
on cost-effectiveness of a scenario of administering a
booster vaccine (one dose) to 50% of females originally
vaccinated was also explored.
In the base case (considering a lifetime duration of
protection, a 100% efficacy, a vaccination coverage of
80% of the eligible population, no cross-protection,
and that screening practices would be unaffected by
vaccination status), it was estimated that an additional
2835 cervical cancer cases and 679 deaths could be
prevented among a cohort of 400,000, at an incremental
cost per QALY gained of €10,530.
One hundred and twenty girls
needed to be vaccinated to prevent
one case of cervical cancer. Costeffectiveness
is sensitive to a
duration of protection of less than
20 years and to the discount rate
for costs and benefits
Furthermore, a scenario in which booster vaccination
is provided (after 10 years) for 50% of females originally
vaccinated in order to ensure lifetime protection produced
an increased cost-effectiveness ratio compared
to the base case, primarily due to the impact of the
increased cost of providing a booster. The impact of
different screening strategies, associated or not with
vaccination, was also considered. In this context,
introducing HPV vaccination in association with a
screening program (less or as efficient as the current
one) appeared more efficient than only improving the
existing screening intervention (screening coverage rate
was increased by 20% without HPV vaccination).
To date, there has been limited use of economic
analyses for such decisions in Germany compared to
some other European countries such as the United
Kingdom. However, this might change with German
insurance companies increasing their focus on cost and
forthcoming requirements for economic analyses to be
performed as part of the reviews of new interventions
by the German Health Technology Agency, the Institute
for Quality and Efficiency in Health Care, which advises
the Federal Ministry of Health.
References: 1. FUTURE II Study Group. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med
2007;356:1915-27. 2. Paavonen J et al. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infections with human papillomavirus
types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised controlled trial. Lancet 2007;369:2161-70. 3. Hillemanns P et al.
J Pub Health, in press.
social aspects of hpv infections
5

6
cross-neuTralizaTion
vaccinescorner
Two virus-like particles (VLP)-based prophylactic vaccines
against HPV-16 and -18, which are together
responsible for around 70% of cervical cancer cases
worldwide, have recently become commercially available.
Both products displayed an excellent safety
profile and high efficacy in preventing persistent HPV
infection and high-grade intraepithelial lesions, which
are obligatory precursors of cervical cancer, in large
placebo-controlled clinical studies. Their potential in
reducing cancer, although expected, still needs to be
evaluated in vaccinated populations and will not be
appreciable for at least one to two decades after launch
of the vaccination campaign.
A somewhat unexpected finding of the clinical trials
was that both vaccines also provide
a certain degree of protection against
other HPV types, particularly -31 and
-45 (and possibly some others), which
are genetically related to HPV-16 or
-18. This cross-protection is most
likely based on the induction of a
more promiscuous immune response
directed against different types. As a
consequence, one could theoretically
prevent up to 80% of cervical cancer
cases (a 10% “bonus” on top of the
coverage against HPV-16 and -18),
although complete protection against
all cases would obviously be most
desirable.
Despite the straightforward, yet scientifically
uninventive, approach of
including "x" more high risk types into
a second-generation vaccine, some
researchers are considering modifying
the L1 molecules that make up the VLPs
in such a way that the particle surface
induces more broadly neutralizing
antibodies. This strategy is challenging
since every manipulation of the L1
protein may alter its ability to assemble
into particulate structures. These structures
present the neutralizing epitopes
in a multimeric array –VLPs consist of
360 L1 molecules– which is a prerequisite
for the well-documented high
immunogenicity of the HPV vaccines.
A possible solution is to use pools of
randomly mutagenized L1 genes for
Lutz Gissmann
Division Genome Modifications and
Carcinogenesis. German Cancer Research
Center. Heidelberg, Germany
the direct (“genetic”) immunization of mice. Only those
molecules that are still assembly-competent will be
able to induce an immune response, which then has to
be tested for cross-neutralization. The pools used to
immunize a positive animal need to be isolated and the
immunization repeated until a L1 clone with the desired
properties has been identified. Only at this point would
it be possible to generate particles from this clone to
verify its immunogenic properties. The biggest hurdle
for this project is proving to be the extremely large
numbers of mice needed for this set of experiments,
which triggers very high costs that are impossible to
cover in an academic setting.
Another road that is being followed by several investigators
is the use of the minor
structural protein L2. This protein is
not required for VLP formation –and
indeed is not part of the existing
vaccines– but appears to be required
to generate infectious virus particles
during the natural life cycle. In the
context of the virus particle, L2 is
not immunogenic, possibly due to
the low copy number per particle.
However, when used as the isolated
protein, L2 or specific L2-derived
epitopes (e.g., of HPV-16) induce
antibodies that neutralize infection
by other HPV types as well. The
major drawback encountered to date
is that the titers induced by L2 are
at least 1000-fold lower than when
L1 VLPs are used for immunization.
Possible solutions to this problem
that are being pursued by different
laboratories are to multimerize the
immunogenic epitopes or to engineer
them into the surface loops
of L1 VLPs, thus presenting them in
the context of the highly symmetric
Whereas the major structural protein L1 is
sufficient for the formation of VLPs, the active
component of the current prophylactic HPV
vaccines, the minor protein L2 is required to
generate infectious virus particles. Recent data
obtained from cryo-EM-based computerized
reconstructions demonstrate that up to 72 copies
of L2 can be incorporated per particle. The model
shows the distribution of L2 molecules (in red)
on the outside (above) and within a virus particle
(below). Reproduced from Buck CB et al. J Virol
2008;82:5190-7.
array. Both strategies have improved
the L2-specific immunogenicity,
albeit not yet to a satisfactory level.
It is now hoped that with the introduction
of rationally designed and
highly efficient adjuvants, the antibody
titers can be further increased
to the point where they may become
clinically relevant.

p16 ink4a as a biomarker for
differenTiaTing rePlicaTing and
Transforming HigH risk HPV
infecTions: THe THeoreTical concePT
and iTs PoTenTial diagnosTic imPacT
Carcinogenesis is regarded as the consequence
of “persistent high risk HPV (HR
HPV) infections” that last for more than 6
months. 1 The term “persistent infection”
is, however, only arbitrarily defined and
does not include any functional or mechanistic
criteria that refer to the molecular
events required for HPV-triggered carcinogenesis.
Although this term is now
widely used, there is a substantial need
to more clearly differentiate the clinically
relevant stages of HR HPV infections on
a molecular basis.
Viral gene expression during an initial HPV
infection follows a distinct pattern that is
strictly related to the differentiation stage
of the squamous epithelial host cells, 2
which permits major viral gene expression
and replication only in terminally
differentiated cells of the intermediate
and superficial cell layers. This stage may
be referred to as “replicating infection”.
However, if control of the viral genes in
the basal cells is lost, the viral oncogenes
E6 and E7 become strongly expressed in
HPV replication
replicating
regulated
normal CIN 1 / LSIL
Brown stain: p16 INK4a
replication-competent basal and parabasal
cells. 3 This triggers chromosomal
instability and may cause transformation
of the affected cells. This latter stage
may thus be referred to as “transforming
infection”. Integration of the viral genome
was initially thought to be responsible
for the sudden deregulation of the viral
gene expression pattern. 4 However, the
observation that integration occurs rather
late in the progression process of high
grade dysplasia, 5 is often undetectable
even in established invasive cancers, 6 and
clearly follows the induction of chromosomal
instability and aneuploidy 7 strongly
suggests that molecular mechanisms
other than integration of viral genomes,
which trigger the deregulated E6-E7 gene
expression profile earlier during the progression
from replicating to transforming
infections, still remain to be discovered.
In comparison to the replicating infections
that may occur in many epithelial sites in
men and women, these transforming infections
primarily occur in epithelial cells at
From the Laboratory
/ transforming HPV infection
E6 / E7 off and p16 INK4a negative E6 / E7 on and p16 INK4a positive
deregulated HPV gene expression
Episomal HPV genomes
CIN 2/3 HSIL CIS /cancer
Integrated HPV genomes
Magnus von Knebel Doeberitz
Department of Applied Tumor Biology.
Institute of Pathology, University of Heidelberg,
Germany.
the uterine transformation zone.
Recent research has shown that the shift
from replicating to transforming infection
is accompanied by massive overexpression
of the cyclin-dependent kinase inhibitor
p16 INK4a (Figure 1). Several lines of
evidence support the notion that interference
of the E7 protein with the pRB-E2F
complex results in undamped expression
of p16 INK4a . Although high p16 INK4a
levels would be lethal for normal cells or
those transformed by other molecular
mechanisms, they are well tolerated by
HPV-transformed cells due to the E7-mediated
short-cut of the tumor-suppressive
pRB pathway.
By staining for p16 INK4a it became possible,
for the first time, to reproducibly visualize
cells that have acquired the transforming
mode of viral gene expression and are thus
at substantially higher risk of neoplastic
progression compared to HPV-infected
cells that still retain their replicating
gene expression pattern and to apply
this technique as a routine diagnostic
Figure 1:
Schematic representation of the
progression from replicating HPV
infections to transforming HPV
infections. During the replicating
infection viral E6 and E7 gene
expression is self-limited in the
basal cells and does not lead to
overexpression of 16 INK4a . However,
it may trigger replication in the
more superficial cells and result in
koilocytosis. Once this control is
lost, E6-E7 gene expression may
occur in the basal cells. This triggers
overexpression of p16 INK4a and
may result in transformation. Since
in the early stages of these transforming
infections differentiation
of the epithelium is still retained,
these lesions still may replicate the
virus as indicated by koilocytosis of
cells above p16 INK4a positive cells in
the basal and parabasal layer.
CIN: Cervical intraepithelial
neoplasia.
HSIL: High squamous intraepithelial
lesion.
CIS: Carcinoma in situ.
7

From the Laboratory
procedure. 8 A series of studies in which the progression
risk of cervical intraepithelial neoplasia (CIN) of grade 1
lesions that were either p16 INK4a -negative or –positive was
analyzed strongly confirmed this notion. 9-11
Given the low reproducibility of
the histopathologic diagnosis,
particularly of CIN2, replacement of
the morphology-based classification
of CIN1, 2 and 3 lesions by a twotiered
novel classification system that
distinguishes only the replicating
and transforming mode of high
risk HPV (HR HPV) infection based
on the simple detection of p16 INK4a
overexpression may be considered.
This approach is likely to increase the diagnostic accuracy
of both histo- and cyto-pathology. Furthermore, it is
in line with the concept of the Bethesda classification
system, which is also based on a two-tiered approach to
differentiate low- and high-grade lesions. 12
The use of p16 INK4a as a biomarker in cytology leads to a
substantial reduction in the number of ambiguous cytology
results. 13,14 Furthermore, the sensitivity for detecting
high-grade disease was found to be in the range of HPV
tests, although with substantially higher specificity. 15
Finally, the biochemical detection of p16 INK4a in cervical
swabs 16 may allow for the development of easy applicable
“point-of-care systems” that may be used either as a
stand-alone test or in combination with HPV testing or
cytology.
Diagnostic algorithms that rely on the molecular distinction
of replicating and transforming HPV infections are,
in principal, novel and may substantially change current
clinical practice. It is thus necessary to define exact
standards and protocols and to carefully evaluate their
full clinical impact in large trials. However, they appear
to have the potential to overcome many of the current
limitations in the diagnosis and screening of HPV-induced
disease.
References: 1. Woodman CB, Collins SI, Young LS. Nat Rev Cancer 2007;7:11-
22. 2. Longworth MS, Laimins LA. Microbiol Mol Biol Rev 2004;68:362-372. 3.
Munger K et al. J Virol 2004;78:11451-11460. 4. Pett MR. Proc Natl Acad Sci U.
S. A 2006;103:3822-3827. 5. Klaes R et al. Cancer Res 1999;59:6132-6136. 6.
Vinokurova S et al. Cancer Res 2008;68:307-313. 7. Melsheimer P et al. Clin Cancer
Res 2004;10:3059-3063. 8. von Knebel Doeberitz M. Eur J Cancer 2002;38:2229-
2242. 9. Kalof AN, Cooper K. Adv Anat Pathol 2006;13:190-194. 10. Negri G et al.
Virchows Arch 2004;445:616-620. 11. Wang SS et al. Cancer Epidemiol Biomarkers
Prev 2004;13:1355-1360. 12. Solomon D et al. JAMA 2002;287:2114-2119. 13.
Carozzi FM. Coll Antropol 2007;31 (Suppl 2): 103-106. 14. Wentzensen N et al.
Cancer 2007;111: 58-66. 15. Szarewski A et al. Cancer Epidemiol Biomarkers Prev
2008;17:3033-3042. 16. Ding L et al. Mol Diagn Ther 2008;12:395-400.
8
monog
HPV-dna inTegraTion in
carcinogenesis does noT
increased leVels of Vira
One factor considered to be of key
importance for the progression of cervical
intraepithelial neoplasia (CIN) to invasive
cervical cancer is integration of HPV into
the host genome. Hopman and co-workers
have shown by in situ hybridization that
88% of CIN2/3 associated with microinvasive
carcinoma contained integrated viral
DNA. In contrast, the characteristic punctuate
signal of integrated viral DNA was
only observed in 29% of solitary CIN2/3. 1
High integration frequencies in cervical
cancer have also been observed in several
other studies in which different techniques
for the detection of integrated viral DNA
(DIPS, E6/E2 ratios) or integrate-derived
viral transcripts (APOT) were used. 2-5
Recent results indicate that viral
integration occurs in up to 88% of
high squamous intraepithelial lesions
(HSIL) associated with micro invasion
as compared to 29% of solitary HSIL.
Integration frequencies close to 90%
have been reported in cervical cancer
but vary strongly according to HPV type.
To date, it is still a matter of debate
whether HPV-DNA integration precedes
E6/E7-induced genetic instability or is a
consequence thereof. In an in vitro study,
acquisition of high levels of genomic
instability in cervical keratinocytes in monolayer
culture occurred after integration of
HPV-16. 6 On the other hand, Melsheimer
and colleagues have shown that in CIN
and cervical carcinomas, aneuploidization
precedes integration of HPV-DNA in the
progression of cervical dysplasia. 7 However,
irrespective of a temporal association
between integration and genetic instability,
the current concept of cervical carcinogenesis
suggests that integration provides the
cell with a selective growth advantage. 8,9
This is in line with the observation that
the viral integration site is unique for each
tumour and remains unaltered during

aph
THe Process of cerVical
necessarily resulT in
l oncogene TranscriPTs
different stages of clonal expansion.
9,10 Several in vitro studies have
indicated that this growth advantage
results from an enhanced
expression of HPV E6 and E7:
HPV integration in W12 cells was
associated with an increase in
levels of detectable E7 protein,
and maximal E7 levels correlated
with the acquisition of structural
chromosomal abnormalities. 6,11
High oncogene levels have been
attributed to the functional loss
of the viral E2 gene product which
acts as an intrinsic repressor of
E6/E7 expression. 12 Moreover,
viral-cellular fusion transcripts are
devoid of the instability elements
located in the 3′-region of the viral
mRNA and may thus have a longer
half-life. 13,14 On the basis of these
experimental data, it is generally
assumed that integration invariably
results in high levels of viral
Relative transcript levels
(plasmid equivalents in 10ng RNA)
oncogene expression. However, does this
model also apply for cervical carcinogenesis?
We have recently analysed the levels
of viral transcripts in 83 HPV-16-positive
CIN and cervical carcinomas (CxCa) in
relation to the physical state of the viral
genome. 15 Using the APOT assay, integratederived
transcripts were only detected
in 3/28 (11%) CIN
and 28/55 (51%)
carcinomas. The
remaining biopsies
contained either
episome-derived
transcripts only or
both mRNA species.
SybrGreen reverse transcriptase
polymerase chain reaction (RT-PCR) assays
were used to quantify viral gene expression
for i) all transcripts initiated from
p97 (E6all); ii) full length E6; iii) E6*I; and
iv) E5 transcripts. As expected, biopsies
with integrate-derived transcripts only
generally lacked E5-specific mRNA (Figure
1,000,000
100,000
10,000
1). The reason for this is that integration
frequently disrupts the viral genome within
the E1 or E2 coding sequences, thereby
uncoupling the promoter p97 from the
E5 open reading frame (ORF). The finding
that the levels of oncogene transcripts
(E6all) in biopsy material were highly
variable but showed similar median values
irrespective of
histopathological
grading and physical
state of the
viral genome is of
particular interest
(Figure 1). These
data strongly suggest
that HPV integration per se does not
invariably result in an increase in oncogene
expression. The highly variable expression
levels of oncogene transcripts observed in
carcinomas are also in line with the results
of Rosty and co-workers 16 but appear to
contradict the data obtained in several
other studies. 17-21 The most plausible
Highly variable levels of viral
oncogene expression are
characteristic of CIN and cervical
cancer and are independent of
histological grading and the
physical state of the viral genome.
Matthias Dürst and Norman Häfner
*Department of Gynaecology, Friedrich-Schiller-University Jena,
Germany.
explanation for these differences
is the sampling strategy used
(biopsies versus cervical scrapes).
Exfoliated cells have the advantage
that they can be obtained
easily and are clearly ideal in a
diagnostic setting, although the
results do not reflect the overall
quantity of the RNA nor do they
represent the diversity of viral
mRNA species in the underlying
lesions. 22
In summary, highly variable levels
of viral oncogene expression are
characteristic for CIN and CxCa
and are independent of histological
grading and the physical
state of the viral genome. These
findings support the hypothesis
that HPV integration primarily
ensures constitutive expression of
the viral oncogenes. The minimal
levels of viral oncogene expression
required to initiate carcinogenesis
and to sustain the transformed phenotype
of the cell remain unknown and could be
tumour-specific. Moreover, other consequences
of integration, such as insertional
mutagenesis and/or effects on the transcriptome,
should also be considered and
should be analysed systematically. 10
E6all
E5
Episomes
n=39
Episomes+integrates
n=13
Integrates
n=31
Figure 1
Relative copy number of HPV-16 oncogene transcripts (E6all) in biopsy
material from CIN2/3 and cervical carcinomas harbouring viral episomes,
co-existing episomes and integrates, or integrates only. Note that the median
values of the oncogene transcripts are similar irrespective of the physical
state of the viral genome. The very low levels of E5 are characteristic for
integrated HPV-DNA since this part of the genome is frequently uncoupled
from the viral promoter.
References: 1. Hopman AH et al. Int J Cancer 2005;115:419-
28. 2. De Marco L et al. J Clin Virol 2007;38:7-13. 3. Klaes
R et al. Cancer Res 1999;59:6132-6. 4. Luft F et al. Int J
Cancer 2001;92:9-17. 5. Peitsaro P et al. J Clin Microbiol
2002;40:886-91. 6. Pett MR et al. Cancer Res 2004;4:1359-
68. 7. Melsheimer P et al. Clin Cancer Res 2004;10:3059-63.
8. Duensing S, Munger K. Int J Cancer 2004;109:157-62. 9.
Wentzensen N et al. Cancer Res 2004;64:3878-84. 10. Kraus
I et al. Cancer Res 2008;68:2514-22. 11. Alazawi W et al.
Cancer Res 2002;62:6959-65. 12. Romanczuk H, Howley PM.
Proc Natl Acad Sci USA 1992;89:3159-63. 13. Jeon S et al.
J Virol 1995;69:2989-97. 14. Jeon S, Lambert PF. Proc Natl
Acad Sci U S A 1995;92:1654-8. 15. Häfner N et al. Oncogene
2008;27:1610-7. 16. Rosty C et al. Oncogene 2005;24:7094-
104. 17. Andersson S et al. Int J Oncol 2006;29:705-11. 18.
Falcinelli C et al. J Med Virol 1993;40:261-5. 19. Molden T et
al. Cancer Epidemiol Biomarkers Prev 2005;14:367-72. 20.
Riethdorf S et al. Int J Gynecol Pathol 2001;20:177-85. 21.
Sotlar K. J Med Virol 2004;74:107-16. 22. Middleton K et al.
J Virol 2003;77:10186-201.
9

Research in Progress
HPV in skin
cancer
The carcinogenicity of HPV-5 and HPV-8 in
the skin is fully acknowledged in patients
with epidermodysplasia verruciformis
(EV), although the role of EV-HPV (genus
beta) in the general population is less
clear. These viruses cause clinically unapparent
infections in almost everyone from
very early on in life, whereas cutaneous
squamous cell carcinomas (SCC) rarely
arise before the age of 65. The quantity
of beta-HPV DNA in skin cancers is usually
very low. 1
During the past five years, research groups
in Germany have expended a great deal
of effort on developing a methodology
for beta-HPV-specific DNA and antibody
detection.
For example, quantitative real-time
polymerase chain reactions (PCRs) have
been developed 2 and serology has been
based on high-throughput multiplex fluorescent
bead-based assays. 3 Furthermore,
transgenic mouse models have been
established for HPV-8 and HPV-20. 4,5
10
Tumor development in HPV-8-
E2-transgenic mice (FVB/N)
caused by UV irradiation with
10 J/cm2 UVA and 1 J/cm2
UVB. 1.5 x 2 x 0.7 centimeters
exophytic, rapidly growing tumor,
which developed within 3 weeks
in the area of a preexisting,
UV-induced ulcus.
Histology reveals epidermis with
superficial erosion and disturbed
stratification, beneath sharply
demarcated spindle-cell tumor
with strong cytological atypia
and atypical mitosis.
The impact of continuous person-toperson
contact within families on an
individual’s beta-HPV type spectrum
has been studied in serial skin swabs
from parents and children. 6 HPV type
multiplicities were found to vary widely
between different families but only slightly
between family members. More than 75%
of the HPV types in babies were also
detected in their parents, thus indicating
that cutaneous HPVs are mainly transmitted
by close contact between family
members. Of the HPV types detected
throughout the study, 24% persisted for
at least 9 months in adults and 11% in
children. Interestingly, about half of the
HPV types found to persist in one of the
parents occurred less frequently or only
sporadically in the spouse, in other words
even regular exposure to cutaneous HPV
does not necessarily lead to a persistent
infection. This may point to type-specific
susceptibilities of different individuals.
Despite very early exposure, antibodies
to the capsid protein L1 of beta-HPV
are rare in children but increase with age
and reach a prevalence of about 35%
later in life. 7 Seroconversion seems to be
extremely slow, possibly due to low-level
viral replication, which is reflected by very
low copy numbers of beta-HPV DNA in
the general population. 2 Weakening of the
immune system and increased sun exposure
later in life may lead to higher viral
loads and higher
seroprevalences. 7
E V p a t i e n t s
were recently reevaluated
using
newly developed
qualitative and
quantitative PCRbased
methods,
in situ hybridization
techniques and serological assays. 8
A multiplicity of beta-HPV genotypes was
detectable in skin samples and plucked
eyebrow hairs. A high intrapatient concordance
for specific types was noted
Case-control studies showed
an increased skin cancer risk
related to beta-HPV infection
(odds ratios 1.5 - 4.4).
The carcinogenic potential of
HPV-8 has been documented
in transgenic mice.
between hair bulbs and skin biopsies,
and antibodies against 16 beta-HPV were
significantly more prevalent and showed
higher titers than in the general population.
More active viral replication in EV
patients was underlined by much higher
viral loads and by strong in situ hybridization
signals in many cell nuclei of an SCC
in contrast to few HPV-positive nuclei in
an SCC of a non-EV patient. 2
German laboratories have participated
in several case-control studies which
have provided some evidence for an
increased risk of cutaneous SCC related
to beta-HPV infection [odds ratios (OR)
in the range 1.5-4.4]. 9-12 These studies
do not consistently point toward specific
high risk beta-HPV types but have shown
significant OR for DNA or antibodies of
any beta-HPV or beta-HPV species 2.
A study of anti-beta-HPV-L1 antibodies in
cutaneous SCC patients, whose plasma
was collected prior to diagnosis, revealed
no statistically significant differences in
comparison to controls. However, consistent
with previous work, the prevalence
of antibodies against many beta-HPVs,
particularly against HPV-8 and HPV-92,
was higher among subjects who had
already developed SCC at blood collection.
13 This may indicate that “danger”
signals related to tumor growth are
important to enhance the slow immune
response to beta-HPV.
It is highly interesting
that a common
polymorphism in
the EV-susceptibility
gene EVER2
appeared to be
associated with
beta-HPV seropositivity,
specifically
HPV-5 or HPV-8
seropositivity, and SCC risk. 14 This opens
perspectives of a role for EV-susceptibility
gene products in the pathogenesis of skin
cancer in the general population.
The carcinogenic potential of HPV-8 has

een documented in transgenic mice
with the early viral genes under control
of the human keratin 14 promoter. These
animals spontaneously develop skin papillomas,
epidermal dysplasia, and in 6% of
cases SCC, without any further treatment
with physical or chemical carcinogens. 4
Interestingly, skin cancer also developed
in mice expressing only the E2 gene of
HPV-8. 15 The rate of tumor formation in
three transgenic lines was found to correlate
with the different E2-mRNA levels.
HPV IN
100 Slides Based
Reference: 1. De Sanjosé S
et al. Worldwide prevalence and
genotype distribution of cervical
HPV DNA in 157,879 women with
normal cytology: a meta-analysis
of 78 studies. Lancet Infect Dis
2007;7:453-59.
Artwork Laia Bruni: WHO/
ICO HPV Information Centre on
HPV and Cervical Cancer.
Blue: Overall HPV-DNA
prevalence
Red: HPV-16 specific
prevalence
Orange: HPV-18 specific
prevalence
Light green: HPVs
other than -16 and -18
specific prevalence
on the published literature, a metaanalysis
was made to provide overall HPV
prevalence estimates in a composite
population of 157,879 women with normal
cytology. These were largely identified in
routine screening programs and were
grouped into five geographical regions. 1
The global estimated HPV-DNA prevalence
was of 10%, and included mostly
oncogenic HPV types. The prevalence
ranges signal Africa as a particularly high
risk region and Europe as a relatively low
Herbert Pfister
Institute of Virology, School of Medical Sciences,
Most lesions initially presented infundibular
hyperplasia and acanthosis combined
with low-grade dysplasia. Severe dysplasia
occurred in 6% of cases. Two carcinomas
revealed a sharply demarcated spindlecell
component, which is reminiscent of a
subset of highly aggressive skin cancers in
immunosuppressed transplant recipients.
Tumor development could be dramatically
enhanced by a single solar-simulated
irradiation with UVA and UVB.
University of Cologne, Germany.
risk region. Among oncogenic types, the
most prevalent ones were HPV-16, -18,
-31, -58, -52, -33, -51, -35, -45, -56, and
-39, with HPV-16 being the dominant type
everywhere (prevalence ranking from
2.3% in Europe to 3.5% in North America)
. There were small variations in the
prevalence and ranking of the remaining
HPV types. Interestingly, these highly
prevalent types also comprise the eight
most important types found in cervical
cancer cases (HPV-16, -18, -33, -45, -31,
References:
1. Pfister H. J Natl Cancer Inst Monogr 2003;31:52-6. 2.
Weissenborn SJ et al. J Invest Dermatol 2005;125(1):93-
7. 3. Waterboer T et al. Clin Chem 2005;51(10):1845-53.
4. Schaper ID et al. Cancer Res 2005;65(4):1394-1400.
5. Michel A et al. J Virol 2006;80(22):11153-64. 6. Weissenborn
SJ et al. J Virol 2009;83(2):811-6. 7. Michael KM
et al. PLoS Pathog 2008;4(6):e1000091. 8. Dell'Oste V et
al. J Invest Dermatol 2008. 9. Karagas MR et al. J Natl
Cancer Inst 2006;98(6):389-95. 10. Waterboer T et al.
Br J Dermatol 2008;159(2):457-9. 11. Forslund O et al.
J Infect Dis 2007;196(6):876-83. 12. Asgari MM et al. J
Invest Dermatol 2008;128(6):1409-17. 13. Casabonne D
et al. Int J Cancer 2007;121(8):1862-8. 14. Patel AS et al.
Int J Cancer 2008;122(10):2377-9. 15. Pfefferle R et al. J
Invest Dermatol 2008;128(9):2310-5.
HPV dna PreValence among women wiTH normal cyTology and HPV
TyPe-sPecific PreValence of THe fiVe mosT frequenT TyPes in eacH
of fiVe world regions: liTeraTure reView
-58, -52 and -35), where HPV-16 accounts
for some 50% of the types and HPV-18 for
some 20% according to similar studies
and literature reviews. The analyses
strongly suggest the biological advantage
of some HPV types (namely HPV-16)
to transmit and persist (as reflected
by the dominant prevalence in women
with normal cytology) and to progress
to cervical cancer (as reflected by their
priority ranking and relative enrichment
in cervical cancer cases).
WHO / ICO Information Centre on HPV and Cervical Cancer (www.who.int/hpvcentre)
11

hpv in screening and triage
12
beyond sTudies:
HPV TesTing in rouTine cerVical cancer screening
Wolfsburg was the first place in Germany where routine
Pap smear screening was introduced and nowadays is
the first city in Europe to successfully run a local primary
screening program for cervical cancer based on HPV
testing and Pap smear. When the Wolfsburg Cervical
Cancer Prevention Project started in February 2006 it
raised a lot of fears amongst gynaecologists throughout
Germany. For women aged 20 or older, an annual visit to
a private gynaecological practice is recommended and
reimbursed by the German health insurance companies.
Apart from pelvic examination, exclusion of infectious
diseases, breast examination and Pap smear, age-specific
counselling (e.g., menopausal problems, contraception)
and general health advice are part of this health checkup.
Officials were afraid that any change in the cervical
cancer screening program would automatically result in
a significant loss of patients. Furthermore, many critics
expected a wave of panic among women with positive
HPV tests. However, nothing of the sort happened in
Wolfsburg. Participation in the Wolfsburg project is high,
with more than 85% of the target population being
screened within the first 30 months. At the same time,
attendance for the annual examination increased in 2006,
the first year of the project, and returned to average rates
WOLFSBuRG SCReeNING PROjeCT
n= 17,087 women 30+ years
Screening Result Final Diagnosis
Colposcopy CIN3+
Cyto+/HPV- 23 0
HPV+/Cyto- 286 52
HPV+/Cyto+ 124 52
Table 1. Only women with persitently positive Hybrid
Capture ® 2 (HC2 ® , Qiagen Gaithersburg, Inc., MD, USA,
previously Digene Corporation) or positive Pap smears
are transferred to colposcopy after 1 year. CIN: Cervical
intraepithelial neoplasia.
in 2007/2008. More
than 99% of women
who attended one
of the participating
gynaecologists in
private practice and
fulfilled the inclusion
criteria (aged
30 years or older,
no history of hysterectomy,
member
of Deutsche BKK)
decided to join
the new routine
program.
Within the first three years of the
project, out of a screened population of
17,000 women 2.4% were transferred
for colposcopy and 1.1% underwent
invasive treatment. exactly 50% of the
104 cervical intraepithelial neoplasia of
grade 3 (CIN3)/cancer cases identified
had normal Pap smears at recruitment
and were only detected because of
positive HC2 ® results (Table 1).
This is one out of 31 cases with normal
Pap smears at months 0, 6 and 12
but positive HPV tests and cervical
intraepithelial neoplasia of grade 3
(CIN3), adenocarcinoma in situ (ACIS)
or invasive cancer on colposcopy at
month 12-15.
Figure 1
Figure 2
Figure 1 shows the colposcopy picture after application of
acetic acid. Because of atypical vessels and dense greyish
epithelium the lesion was classified as high-grade.
Figure 2 shows the histology picture showing an adenocarcinoma
in situ.
Case No. 1 Age: 45 years
29/01/2007 Pap II HPV positive
11/05/2007 Pap II
25/07/2007 Pap II
03/03/2008 Pap II HPV positive
21/04/2008 Colpo-Clinic Adeno-Ca in situ
Table 2. HPV persistency prompted colposcopy in the presence of repeated
normal cytology
The telephone hotline installed to calm fears among
worried participants with positive results was rarely
used, and there was no “HPV-induced hysteria”.
One key to the program’s success was the involvement

Alexander Luyten
Frauenklinik im Klinikum Wolfsburg, Germany.
of all gynaecologists in all steps during the development
of the project, including patient information and the
definition of patient pathways. Deutsche BKK is the
health insurer for roughly half of the population in Wolfsburg
and was the other driving force behind the project
as its management agreed to reimburse a recruitment
fee, data transmission, HPV testing and colposcopy.
This investment is recovered by the reduced numbers
of Pap smears, examinations and treatments. Screening
intervals for women with negative Hybrid Capture ® 2
(HC2 ® Qiagen Gaithersburg, Inc., MD, USA, previously
Digene Corporation) tests and normal Pap smears are
extended to five years instead of the annual Pap smear
in the German routine screening program. Triaging tests
as well as referrals for colposcopy are supervised by a
central data-management unit.
Even among the 52 cases with positive Pap smears
at recruitment, 43 had an earlier diagnosis because
CONSORTIUM
Monika Hampl, gynaecology, vulvar cancer
Düsseldorf
Herbert Pfister, virology, skin cancer
Sigrun Smola, virology
Thomas Löning, pathology
Peter Hillemanns, gynaecology
Maria Blettner, Stefanie Klug, epidemiology
Thomas Iftner, virology, screening
Köln
THe german neTwork of HPV
and cancer researcH: THe HPV
managemenT forum
Frankfurt
Mainz
Heidelberg
Tübingen
Hamburg
Hannover
emmendingen
Peter Schneede, urology
Jena
Rostock
Gerd Gross, dermatology
BeRlin
Wolfsburg
K. ulrich Petry, gynaecology, screening
Hans Ikenberg, gynaecology
Matthias Dürst, tumour virology
Harald zur Hausen, virology
Lutz Gissmann, virology
Magnus von Knebel-Doeberitz, molecular biology
Michael Pawlita, molecular biology
Karl ulrich Petry
Frauenklinik im Klinikum Wolfsburg, Germany.
atypical squamous cells of undetermined significance
(ASCUS) and low squamous intraepithelial lesion (LSIL)
cases with positive HC2 ® tests were immediately transferred
for colposcopy. Some other health insurers may
join this project, which has already been extended to
neighbouring areas. The Wolfsburg project demonstrates
that HPV screening is feasible, improves the quality of
cervical cancer prevention and is highly accepted among
gynaecologists and the female population.
The biggest remaining problem within the project is the
best management of HC2 ® positive women with normal
cytology results. The current triaging with repeat Pap
smear at month six only detects a minority of cases,
and three invasive cancer cases were only diagnosed
because of persistent positive HC2 ® results after twelve
months. Hopefully, adjunct scientific trials will lead to
better tools that allow an earlier identification of CIN3+
cases.
The HPV-Management Forum
is a working group of the
German Paul-Ehrlich-Gesellschaft
für Chemoptherapie. It
consists of a multidisciplinary
consortium of individuals
and centers experienced in
basic and clinical HPV-related
sciences.
Achim Schneider, gynaecology, oncology
Andreas Kaufmann, immunology
13

comParison of THe Performance of differenT HPV geno-
TyPing meTHods for deTecTing geniTal HPV TyPes
S. J. Klug, A. Molijn, B. Schopp, B. Holz, A. Iftner, W. Quint, J. F. Snijders, K. U. Petry, Kjaer S.
Kruger, C. Munk, and T. Iftner. J Med Virol 2008;80:1264-74.
This study evaluates the performance of the three L1-polymerase chain reaction
(PCR)-based assays PGMY09/11 LBA, HPV-DNA Chip and SPF LiPA, and an E1 consensus
PCR followed by cycle sequencing (E1-PCR). The four tests were evaluated
crosswise using 824 cervical smears pre-tested by Hybrid Capture ® 2 (HC2 ® , Qiagen
Gaithersburg, Inc., MD, USA, previously Digene Corporation). Differences were noted
in the sensitivity and range for specific HPV types. HPV-16 was the most prevalent
type detected by all tests except for SPF-10 LiPa, which detected HPV-31 more
often. Calculated kappa values were higher for high risk type positivity (k=0.31–0.61)
and best for recognition of HPV-16 (k=0.53–0.72). The analytical sensitivity of the
tests ranged between 15% and 97% for individual types and specificity was highly
dependent on which test system was used as ‘‘gold standard’’ for the analysis.
E1-PCR, PGMY09/11 LBA and SPF-10 LiPA had a high clinical sensitivity (>95%) for
the detection of cervical intraepithelial neoplasia (CIN)2+, whereas the HPV-DNA
Chip reached only 84.1%.
new asPecTs of VulVar cancer: cHanges in localizaTion
and age of onseT
M. Hampl, S. Deckers-Figiel, J. A. Hampl, D. Rein, and H. G. Bender. Gynecol Oncol 2008;109:340-5.
Although the incidence of invasive vulvar cancer has been reported to be stable or
only minimally increased, the results of this study, conducted at one university hospital
unit in Germany, show that the number of cases has doubled within the last three
decades, with a nearly fourfold increase in younger patients (+372%) due to high risk
HPV infection. The total increase was 192%. Two thirds of the tumors in women aged

VulVar cancer in THe anTerior fourcHeTTe
in a young woman
50 years and 25% under 40 years of age.
Small non-invasive, microinvasive, and
invasive lesions in the non-keratinized
region between the clitoris and the
urethra are increasingly being seen in
Germany. 2 In our patient cohort, 38 out
VuLVar CaNCEr
uFK Duesseldorf 1980-2007
Table 1
january 1980
February 1989
of 102 women presented with lesions in
the anterior fourchette (37%; 1998-2007).
The tumors in this area were high risk HPV
positive in 61% of cases. These lesions are
sometimes hard to see and are therefore
diagnosed late, at which point they often
Changes in the incidence of vulvar cancers treated at the Department of Obstetrics and Gynecology, University of Duesseldorf,
Germany, between 1980 and 2007
Figure 2
Period
March 1989
April 1998
May 1998
june 2007
Number 53 69 102
< 50 years 6 (11%) 17 (25%) 42 (41%)
> 50 years 47 (89%) 52 (75%) 60 (59%)
Mean age (years) 65.6 63.9 57
Area
between clitoris/urethra 10 (19%) 14 (20%) 38 (37%)
perineum 5 (9%) 14 (20%) 13 (13%)
labia 26 (49%) 21 (30%) 27 (26%)
elsewhere 12 (23%) 20 (29%) 21 (20%)
Figure 1
Tumor location as seen on admission in our case of
a 27-year-old woman.
Acceptable cosmetic result after local tumor resection
(pT1b tumor).
involve the orificium externum of the
urethra (14%) and therefore require partial
urethral resection (in up to 15% of cases).
There are several potential explanations
for this observation. First of all, the epithelium
between the clitoris and urethra is a
non-keratinized squamous cell epithelium.
It therefore seems conceivable that, in
comparison with the transformation
zone on the cervix, the non-keratinized
epithelium in this region is less protected
from viral infection because of this lack of
keratin and might be more susceptible to
the small tears or lesions which favor and
facilitate infection with HPV. The increasing
number of women being infected with high
risk HPV may contribute to this change in
tumor localization. Although our data for
HPV-involvement in vulvar cancer concern
only a relatively low number of women
with HPV-positive lesions (n=18), 11 of
these lesions were located in the anterior
fourchette. To verify these data, we will
perform HPV typing in a larger series of
these tumors.
Whether changes in genital hygiene practices,
such as the use of rougher toilet
paper, also play a role is purely speculative.
Another explanation could be the
trend towards an earlier onset of first
sexual contact in young females, which
often starts with non-penetrative sexual
practices that might be sufficient for this
area to become infected with HPV. This
could result in an early-onset, pre-invasive
neoplasia and subsequent development of
invasive tumors years later.
We conclude that the anterior vulvar
fornix and periclitoral region seems
to be a predisposed location for
tumors in young women and should
therefore be examined thoroughly by
colposcopy and biopsy in the event
of a suspicious lesion.
ReFeReNCeS: 1. Hampl M et al. A case of a pT3 HPV -52 positive vulvar cancer in an 18 year-old woman. Gynecol Oncol 2006;101:530-33. 2. Hampl M et al. New aspects in vulvar
cancer: changes in localization and age of onset. Gynecol Oncol 2008;109:340-45. 3. Jones RW, Baranyai J, Stables S. Trends in squamous cell carcinoma of the vulva: the influence
of VIN. Obstet Gynecol 1997;90:448-52. 4. Judson PL et al. Trends in the incidence of invasive and in situ vulvar carcinoma. Obstet Gynecol 2006;107(5):1018-22
15

Barcelona, Spain
22 nd – 24 th june 2009
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Active Immunotherapeutics
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