Oral Oncology 49 (2013) 937942

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Oral Oncology
j o u r n a l h o m e p a g e : w w w . e l s e v i e r.c o m / l o c a t e / o r a l o n c o l o g y

Lack of evidence of human papillomavirus-induced squamous cell

carcinomas of the oral cavity in southern Germany
Miriam Reuschenbach a,b,, Katinka Kansy c, Kira Garbe a,b, Svetlana Vinokurova a,b,
Christa Flechtenmacher d,e, Csaba Toth d,e, Elena-Sophie Prigge a,b, Oliver C. Thiele c, Siegmar Reinert f,
Jrgen Hoffmann c, Magnus von Knebel Doeberitz a,b, Kolja Freier c
a

Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany
Clinical Cooperation Unit Applied Tumor Biology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 224, 69120 Heidelberg, Germany
c
Department of Oral and Maxillofacial Surgery, University of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany
d
Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany
e
National Center for Tumor Diseases (NCT) Tissue Bank, Im Neuenheimer Feld 221, 69120 Heidelberg, Germany
f
Department of Oral and Maxillofacial Surgery, University of Tbingen, Osianderstr. 2, 72076 Tbingen, Germany
b

a r t i c l e

i n f o

Article history:
Received 13 February 2013
Received in revised form 19 March 2013
Accepted 24 March 2013
Available online 19 April 2013
Keywords:
Oral cavity
Squamous cell cancer
Human papillomavirus
INK4a

p16
Ki-67

s u m m a r y
Objectives: The aim of the present study was to identify HPV-attributable SCC of the oral cavity (OSCC) in
a cohort of patients from southern Germany.
Materials and methods: A sensitive PCR-enzyme immunoassay (EIA) was followed by a more specic
in situ hybridization (ISH) to detect high risk human papillomavirus (HPV). An immunohistochemical
dual-staining for p16INK4a and the proliferation marker Ki-67 was used to assess whether co-expression
of p16INK4a/Ki-67 is a better surrogate marker for HPV in OSCC than p16INK4a alone, based on the hypothINK4a
esis that combined p16
and Ki-67 expression might specically discriminate oncogene-induced
INK4a
INK4a expression from cell-cycle arrest-inducing senescence-associated p16
expression.
p16
Results: HPV-DNA by PCREIA could be detected in 25.1% (69/275) of the tumors, but ISH was negative in
all of them. Diffuse p16INK4a overexpression was detected in 11 HPV PCR-positive tumors, but also in 6
HPV PCR-negative tumors. p16INK4a-expressing cells in diffusely positive tumors co-expressed Ki-67, irrespective of the HPV status. Neither the sole HPV status nor combined HPV/p16INK4a status nor the sole
p16INK4a status was signicantly associated with disease free or overall survival, however a trend
towards better overall survival of patients whose tumor expressed p16INK4a in a focal pattern (=p16INK4apositive/ Ki-67-negative cells) compared to no p16INK4a expression (p = 0.09) was observed.
Conclusion: Viral DNA can be detected in some tumors by a sensitive PCR, but absence of ISH signals indicates that the HPV-attributable fraction is smaller than estimated from PCR positivity. p16INK4a/Ki-67 coexpression is detectable in a fraction of OSCC irrespective of the HPV status.
2013 Elsevier Ltd. All rights reserved.

Introduction
High-risk human papillomaviruses (HR-HPVs) are necessary for
the development of virtually all cervical cancers, a substantial proportion of other anogenital cancers, and viral DNA can be detected
1,2
in about 25% of head and neck squamous cell carcinomas. HPV-

Abbreviations: DFS, disease-free survival; EIA, enzyme immunoassay; HPV,

human papillomavirus; HR-HPV, high-risk human papillomavirus; ISH, in situ
hybridization; OS, overall survival; OSCC, squamous cell cancer of the oral cavity;
PCR, polymerase chain reaction; SCC, squamous cell cancer.
Corresponding author at: Department of Applied Tumor Biology, Institute of
Pathology, University of Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg,
Germany. Tel.: +49 6221 56 35591; fax: +49 6221 56 5981.
E-mail address: miriam.reuschenbach@med.uni-heidelberg.de (M. Reuschenbach).
1368-8375/$ - see front matter
2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.oraloncology.2013.03.451

induced malignant transformation requires the overexpression of

the viral oncogenes E6 and E73 and the sole detection of viral
DNA cannot be regarded as evidence for a causal involvement of
HPV in tumor development. Data on HPV-association with oropharyngeal SCC primarily involving the tonsil and base of tongue are
increasingly compelling by demonstrating that viral oncogenes
4,5
and associated biomarkers are overexpressed and that patients
with these likely true HPV-driven tumors have a better prognosis,
highlighting the potential diagnostic relevance of identifying HPVinduced tumors in the head and neck region.68
The evidence for a causal involvement of HPV in SCC development at other sites of the head and neck region, including the oral
cavity, is less clear. Each year, nearly 300,000 new cases of oral
cancer are identied worldwide.9 Tobacco and alcohol are strong
risk factors and genetic alterations, such as affecting CDKN2A are

M. Reuschenbach et al. / Oral Oncology 49 (2013) 937942

2
10

important carcinogenic drivers. HPV DNA has been found in a

percentage of oral SCC (OSCC) with an average of 20.2% (95% condence interval 16.025.2%) in 60 studies on 4195 patients in a recent meta-analysis.11 However, data on the percentage of OSCC
that is causally linked to HPV oncogene expression are scarce
and currently suggest that the fraction of HPV-attributable cancers
might be much smaller than estimated from HPV DNA PCR
12
positiv- ity, although there is no clear evidence yet on which is
the gold standard test to identify HPV-driven O SCC. Detection of
transcrip- tionally active viral oncogenes by HPV E6/E7 mRNA
assays has been used and yielded positive results ranging from
0% in 50 pa- tients from Latin America and Central Europe,13
12
5.9% in 409 pa- tients from the USA
to 14.7% in 68
patients from Japan.14
Similarly, in situ hybridization (ISH) of viral genomes is considered
more specic for relevant HPV than PCR, most likely because the
latter may easily pick up contaminations of HPV genomes that
are not associated with the cancer, however potentially with a lower sensitivity of ISH than DNA and RNA amplication techniques.15,12 Furthermore detection of p16INK4a overexpression is
discussed as a marker for HPV-driven head and neck cancers due
INK4a
to the indirect transcriptional activation of p16
expression
16,17
by the HPV E7 oncoprotein.
However, while for tonsillar canINK4a
cers p16
overexpression has been described strongly associated with HPV and is discussed as a simple diagnostic tool to
identify HPV-associated tonsillar SCC,15 data for OSCC are less clear
and one recent study reports that over 50% of p16INK4a-positive

an enzyme immunoassay (EIA) by binding to streptavidin-coated

microtiter plates (Roche, Mannheim, Germany), hybridization to
a mix of HPV type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66
and 69-specic digoxigenin-labeled probes followed by detection
with alkaline phosphatase-labeled anti-digoxigenin Fab fragments
(Roche, Mannheim, Germany), Alkaline Phosphatase Yellow substrate (Sigma) conversion and measurement of the optical density
(OD) at 405 nm. Samples with OD threefold exceeding background
OD were considered positive. Amplication of beta globin was performed to ensure sufcient DNA integrity.

OSCC are negative for HPV E6/7 expression.12

Ki-67 expression using the CINtec PLUS dual-staining kit (mtm

Laboratories AG, Heidelberg, now Roche Diagnostics), according
to the manufacturers protocol applying some minor modications
for use in histology as described previously.18 The staining is based
INK4a
on two monoclonal antibodies, one directed to human p16
protein (clone E6H4) and the other to human Ki-67 protein

The aim of the present study was to (a) determine the likely
HPV-attributable fraction in a large German cohort of SCC located
in the oral cavity by combining a sensitive PCR followed by a more
specic HPV ISH assay and (b) to apply an immunohistochemical
dual-staining for p16INK4a and the proliferation marker Ki-67 in order to assess whether co-expression of p16INK4a/Ki-67 is a better
INK4a

surrogate marker for HPV in OSCC than p16

alone, based on
the hypothesis that combined p16INK4a and Ki-67 expression
INK4a
might specically discriminate
oncogene-induced
p16
expression
from cell-cycle arrest inducing senescence-associated p16INK4a
expression18 and (c) to evaluate the prognostic impact of the
HPV and p16INK4a/Ki-67 status on patients outcome.
Materials and methods
Patients and tumor material
Included in the study were patients with squamous cell
cancers located in one of the following localizations: oor of
mouth, ante- rior tongue, mandibular alveolar process, buccal
mucosa, maxillary alveolar process or lip mucosa and treated
between 1988 and 2005 at the Departments of Oral and
Maxillofacial Surgery of the Univer- sity Hospital of Tbingen or
Heidelberg, Germany. Formalin-xed, parafn-embedded (FFPE)
pre-therapeutic tumor tissue from the patients was retrieved
from the pathology archives and the tissue bank of the National
Center for Tumor Diseases (NCT), Heidelberg, Germany following
the institutional ethical approvals and patients were excluded
from the study if no material was available. The his- topathologic
diagnosis of SCC was veried by a pathologist in addi- tion to the
previous evaluation for routine diagnostics. Clinical data were
retrieved from the patients hospital les.
Detection and genotyping of human papillomavirus DNA by PCR
DNA was extracted from FFPE tissue sections using the DNeasy
Blood & Tissue Kit by Qiagen, Hilden, Germany. HPV detection was

Detection of HPV by in situ hybridization

In situ hybridization for HPV on FFPE sections was performed
for all HPV GP5+6+-EIA-positive tumors. The GenPoint (Dako,
Glostrup, Denmark) HPV probe set hybridizing DNA from oncogenic types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68
and the GenPoint Detection System (Dako) for visualization
was used according to the manufacturer instructions. Cervical cancer sections were used as positive control and sections were processed in parallel without probe incubation as negative control.
Immunohistochemical staining for p16INK4a/Ki-67
INK4a

FFPE sections from all tumors were analyzed for p16

(clone 274-11 AC3). p16

INK4a

and

is visualized by a horseradish perox-

idase-mediated conversion of 3,3-diaminobenzidine (DAB) chromogen leading to a brown cytoplasmic staining and Ki-67 by
alkaline phosphatase-mediated conversion of Fast Red chromogen
leading to a red nuclear staining. Slides were evaluated according
INK4a

to

p16

expression patterns

(diffuse = continuous

clonal

performed by amplication of a viral consensus L1 sequence using

biotinylated GP5+6+ primers as described previously19 followed by

M. Reuschenbach et al. / Oral Oncology 49 (2013) 937942

expression beginning in the basal and parabasal tumor cell layers

and to a variable extent continuously reaching the remaining tumor areas, focal = expression in cell clusters or single cells, may
be extensive, but does not include clonal expression beginning in
INK4a
basal and parabasal cell layers, negative = no expression of p16
in the tumor cells) as well as the presence of cells co-expressing
p16INK4a and Ki-67.
Statistics

Absolute numbers and frequency distributions are provided for

categorical variables and mean with minimum, maximum and
standard deviation are provided for age (years). Independent samples T test was used to assess differences in patients age between
different groups of categorical variables and Fishers exact test or

Chi-square test was used to assess differences between categorical3

variables. Unknown clinical or histopathologic data are reported as
unknown and excluded from calculations. KaplanMeier analysis
was used to estimate survival times.
Results
Patients and tumors characteristics
Oral SCC from a total of 275 patients were included. Mean age
of the patients at tumor diagnosis was 59.9 years (min 16, max
93, standard deviation 11.3 years), most patients were male
(206/
275, 74.9%) and the most frequent tumor localization was the oor

of mouth (107/275, 38.9%). The majority of the patients underwent

only surgical tumor resection (196/275, 71.3%), followed by 12.7%
(35/275) of the patients receiving surgery and adjuvant radiotherapy. Detailed description of the cohort is shown in Table 1.

continuous pattern (example in Fig. 2A and B). All 17 tumors with

diffuse p16INK4a expression pattern contained cells co-expressing
INK4a
p16
and Ki-67, while this was not observed in any of the 47 tumors with focal p16 expression patterns, where p16 expression appears restricted to the central tumor region and Ki-67 expression

Prevalence of human papillomavirus

Archival tissue from all 275 patients was analyzed for DNA of

to the invasion front (Fig. 2). In 11 samples without p16

INK4a
expression in the tumor, p16
was detectable in adjacent
non-invasive cells.

oncogenic mucosal HPV types by PCR (HPV-EIA). A total of 69

Although diffuse p16INK4a expression correlated with HPV-EIA

INK4a

INK4a

(25.1%) tested positive and was further characterized by ISH for

positivity (p < 0.001) and 11 of the 17 diffusely p16

mucosal high risk HPV. While cervical cancers used as positive controls for HPV ISH regularly displayed nuclear spots in tumor cells

tumors were HPV-EIA-positive, 6 of diffusely p16

-positive tumors were HPV-negative and the majority of HPV-EIA-positive tu-

(Fig. 1A and B), this was not observed in any of the analyzed
HPV-EIA-positive OSCC (Fig. 1C and D).
HPV DNA detection by PCR was not signicantly associated
with patients gender, age, tumor localization, tumor grade or stage
or the year of tumor diagnosis (Table 1).

mors did not diffusely express p16

(Table 2). Focal p16
expression was observed to equal extents in HPV-EIA-positive
and negative tumors (Table 2).

INK4a

Expression patterns of p16

, proliferation (Ki-67 expression) of
p16INK4a-expressing cells and correlation with HPV status
INK4a

Expression of the cell cycle regulator p16

was analyzed
immunohistochemically in conjunction with the proliferation marker Ki-67 applying a dual-staining protocol. The majority of all tumors (211/275, 76.7%) did not express p16INK4a. A diffuse p16INK4a
expression of the entire tumor area was observed in 6.2% (17/275)
of all SCC (example in Fig. 2C and D). Seventeen percent (47/275) of
INK4a
the tumors displayed focal p16
expression of cell clusters of
varying extent, but not reaching the entire tumor area in a diffuse,

-positive

INK4a

INK4a

Prognostic signicance of HPV and p16INK4a status

In order to assess whether tumor HPV DNA presence detected
INK4a
by EIA or tumor HPV DNA presence in diffusely p16
-positive
tumors only was associated with overall (OS) and disease-free survival (DFS), KaplanMeier plots were computed (Fig. 3). The mean
follow-up time of the patients was 37 months (max 183.6, min
0.2). Neither the sole HPV status (OS p = 0.88, DFS p = 0.86) nor
the combined HPV/diffuse p16INK4a expression status (OS
p = 0.80, DFS p = 0.35)) was signicantly associated with overall
and disease-free survival. When evaluating the impact of the
INK4a
p16
expression status only, there was a trend towards better
overall survival of patients whose tumor expressed p16INK4a in a

Table 1
INK4a
Characteristics of the 275 patients in relation to HPV and p16
expression status.
HPV-PCR

p16

Total (n = 275)

Positive (n = 69)

Negative (n = 206)

143
132

52.0
48.0

40
29

58.0
42.0

103
103

50.0
50.0

69
206

25.1
74.9

15
54

21.7
78.3

54
152

Tumor localization
Floor of mouth
Tongue
Mandibular alveole
Buccal mucosa
Maxillar alveole
Lip mucosa

107
60
55
14
23
16

38.9
21.8
20.0
5.1
8.4
5.8

29
16
13
4
2
5

42.0
23.2
18.8
5.8
2.9
7.2

Tumor grade
G1
G2
G3
G4
Unknown

37
151
49
1
37

15.5
63.4
20.6
0.4

9
36
11
0

Tumor stage
T1
T2
T3
T4
Unknown

74
91
14
88
8

27.7
34.1
5.2
33.0

122
18
71
4

56.7
8.4
33.0
1.9

Age
660 years
>60 years
Gender
Female
Male

Nodal stage
N0
N1
N2
N3

INK4a

expression

Diffuse

Focal

Negative

0.268

10
7

58.8
41.2

24
23

51.1
48.9

109
102

51.7
49.5

0.842

26.2
73.8

0.523

2
15

11.8
88.2

15
32

31.9
68.1

52
159

24.6
77.2

0.247

78
44
42
10
21
11

37.9
21.4
20.4
4.9
10.2
5.3

0.539

6
5
3
1
2
0

35.3
29.4
17.6
5.9
11.8
0.0

20
10
5
2
7
3

42.6
21.3
10.6
4.3
14.9
6.4

81
45
47
11
14
13

38.4
21.8
22.3
5.3
6.6
6.3

0.639

13.0
52.2
15.9
0.0

28
115
38
1

13.6
55.8
18.4
0.5

0.948

1
8
6
0

5.9
47.1
35.3
0.0

8
29
7
0

17.0
61.7
14.9
0.0

28
114
36
1

13.3
55.3
17.1
0.5

0.569

16
22
4
24

23.2
31.9
5.8
34.8

58
69
10
64

28.2
33.5
4.9
31.1

0.846

2
5
1
8

11.8
29.4
5.9
47.1

14
16
5
12

29.8
34.0
10.6
25.5

58
70
8
68

27.5
34.0
3.8
33.0

0.320

26
7
18
0

37.7
10.1
26.1
0.0

96
11
53
4

46.6
5.3
25.7
1.9

0.258

11
1
2
0

64.7
5.9
11.8
0.0

28
3
10
0

59.6
6.4
21.3
0.0

83
14
59
4

39.3
6.8
28.0
1.9

0.284

Unknown

60

Figure 1 HPV in situ hybridization of SCC of the uterine cervix as positive control (A 10x objective and B 40x objective) with brown nuclear spots (black arrow) and an
example of a HPV PCREIA-positive SCC of the oral cavity (C 10x objective and D 40x objective) without brown signals indicating absence of viral genomes in the tumor
cells.

INK4a

INK4a

Figure 2 Immunohistochemical dual-staining for p16

and Ki-67 of oral cavity SCC. (A) (10x objective) and (B) (40x objective) show a tumor with focal p16
INK4a
INK4a
expression pattern and absence of cellular co-expression of p16
and Ki-67 (brown p16
expressing cell, white arrow and red Ki-67 expressing cell, black
INK4a
INK4a
arrow). (C) (10x objective) and (D) (40x objective) show a tumor with diffuse p16
expression pattern with cellular co-expression of p16
and Ki-67 (red Ki-67INK4a
expressing nuclei, indicated by black arrow in brown p16
-expressing cells, indicated by white arrow).

Table 2
Numbers of HPV-EIA-negative and positive tumors in relation to p16 expression
status.
p16 Immunohistochemistry

HPV-EIA

Negative

Focal

Diffuse

Negative
Positive

164
47

36
11

6
11

focal pattern (=p16INK4a-positive/Ki-67-negative cells) compared to

INK4a
no (p = 0.09) or diffuse p16
expression (p = 0.25).

Discussion
In the present study a large cohort of patients with SCC located
in the oral cavity from two German university hospitals was investigated for HPV in the tumor tissue by means of different approaches. Although viral DNA was detectable in 25% of the
tumors by PCR using a HPV-EIA and thereby meeting frequencies
11
reported from other studies , no HPV was detectable using an
ISH protocol commonly yielding clear signals in cervical cancers
(10/10 positive in20), its precursors (20/20 high grade dysplasia
20
positive in ), HPV16-associated oropharyngeal cancers (136/146

Figure 3 KaplanMeier curves for overall (AC) and disease-free (DF) survival. A and D: sole HPV-EIA status (OS p = 0.88, DFS p = 0.86); B and E: combined HPV-EIA/diffuse
INK4a
INK4a
INK4a
p16
expression status (HPV-EIA-positive and p16
diffusely positive vs. the remaining tumors) (OS p = 0.80, DFS p = 0.35); C and D: sole p16
expression status
INK4a
INK4a
INK4a
(p16
in a focal pattern (=p16
-positive/Ki-67-negative cells) compared to no (p = 0.09) or diffuse p16
expression (p = 0.25)).

positive in15) and also in all cervical cancers included in each

stain- ing run in our study (example in Fig. 1A and B). Thus the
results provide further evidence that the percentage of HPVinduced OSCC is most likely frequently overestimated when viral
DNA is detected by PCR. Although it has to be recognized that ISH
lacks maximum sensitivity and there have been tumors described
in the literature where viral oncogene expression might suggest
12
HPV-association but ISH was negative (4/24 OSCC in ), ISH has
the advantage that it is a highly specic test to visualize viral
genomes directly in the tumor and can be used on older
archival tumor material where RNA integrity is limited.21
INK4a
For tonsillar SCC immunohistochemical detection of p16
has shown promising performance in diagnosing HPV-associated
cancers15, however the more accurately the anatomic localizations
were stratied in recent studies, the more the relevance of
INK4a
p16
overexpression for SCC in non-oropharyngel sites, such as
the oral cavity, appears less clear.12,22 Our results conrm that
INK4a
INK4a
although the majority of OSCC are p16
-negative, p16
may be found overexpressed also
in HPV-negative tumors
(6/17
diffusely p16INK4a-positive tumors) in
a
pattern
mimicking the
diffuse expression usually seen in
HPVassociated tumors. Whether the
11 HPV PCREIA-positive diffusely p16INK4a-positive tumors

overexpress viral oncogenes could not be determined. However,

absence of ISH signals in all of these tumors argues against a contribution of HPV in the majority of them. One might hypothesize
INK4a
that p16
expression in tumors which are not induced by
HPV might be the consequence of a senescence-like tumor reaction
and that p16INK4a expressing cells in these tumors are therefore
cell cycle arrested (Ki-67-negative). However, our results disprove
INK4a
this hypothesis as p16
-expressing cells
that retained
proliferation capacity (co-expressing p16INK4a and Ki-67) were
found in both, HPV-positive and negative tumors. While lacking
INK4a
cell cycle arrest in p16
-expressing cells in cervical cancers has
been explained by HPV oncogenes that are known to disrupt the
downstream play- ers of p16INK4a (retinoblastoma protein),18 it
may be speculated that other oncogenic stimuli that disrupt this
INK4a
pathway may result in proliferating p16
-expressing cells.
INK4a
Interestingly, patients with focally p16
-expressing OSCC
tended to have a better overall survival than patients with
INK4a
tumors lacking p16
expres- sion (P = 0.09) or diffusely
INK4a
overexpressing p16
(p = 0.25) in the present study. As focally
INK4a
INK4a
p16
-expressing tumors never showed p16
-positive cells
co-expressing Ki-67, which indicates cell cycle arrest, it is
conceivable that these tumors have successfully entered a
senescence state, potentially resulting in slower tumor

growth and better outcome. It will be important in future

prospec- tive studies to conrm this nding.
For oropharyngeal cancers involving the tonsil and base of ton68
gue the prognostic relevance of the HPV status is compelling.
With respect to the prognostic relevance of HPV in OSCC there is
no such clear evidence in the literature, with few studies reporting
a better outcome for HPV-positive patients, 2325 few reporting a
2628
29,30
worse outcome
and others nding no effect.
The HPV
INK4a
PCREIA status and also the combined HPV/p16
status did
not correlate with disease-free and overall survival in our study.
Considering that the result of viral E6/E7 expression in single tu12,4
mors located in the oral cavity described in the literature
indicates HPV-driven cancers, it will be important to evaluate whether
here the HPV status is of prognostic relevance with the
limitation of
such studies that E6/7-positive cancers are
considered as the gold standard for HPV-driven OSCC, which is
unfortunately dif- cult
to prove by additional evidence.
Furthermore different E6/E7 assays are likely
to perform
heterogeneous in terms of their analyt- ical characteristics and
considering the estimated small fraction of HPV-induced oral
cavity cancers large cohorts will have to be ana- lyzed to evaluate
the prognostic relevance.
In summary, we here add further evidence that HPV-attributable cancers in the oral cavity are rarely found although viral
DNA by PCR has been detected in ours and other studies in a larger
percentage in cancerous and also normal epithelium of the oral
31
cavity. Apparently the induction of HPV-driven transformation
occurs particularly at certain anatomic sites, the uterine cervix,
the anal canal and tonsils, all of which are characterized by transition of squamous cell epithelium into glandular or reticular
epithe- lium (tonsillar crypt). While on the one hand these zones
might facilitate access of the virus to basal cells and thereby
increasing the risk for infection in general, there are also results
showing a distinct population of cells with unique morphology
and gene- expression prole at the cervical transformation
zone,32 which might play a role in controlling viral oncogene
expression in these cells.
Acknowledgements
The authors thank Jonathan Drre, Heike Sartor and Bettina
Kast for excellent technical assistance. Author Elena-Sophie Prigge
was supported by the Heinrich F.C. Behr-Stipendium awarded by
the German Cancer Research Center (DKFZ), Heidelberg, Germany.
Parts of the analyzed samples were kindly provided by the Tissue
Bank of the National Center for Tumor Diseases (NCT), Heidelberg,
Germany (Dr. E. Herpel). mtm Laboratories donated antibodies for
the study.
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