Gynecologic Oncology 104 (2007) 721 – 726

www.elsevier.com/locate/ygyno

Predictive value of HPV DNA in lymph nodes in surgically treated cervical

carcinoma patients—A prospective study
Krzysztof Lukaszuk a , Joanna Liss b , Jacek Gulczynski c , Monika Nowaczyk d ,
Miroslaw Nakonieczny a , Mariusz Piatkowski b , Wojciech Sliwinski b,⁎, Marc Baay e ,
Izabela Wozniak a , Bozena Maj b , Mariusz Lukaszuk b
a
Department of Gynaecological Endocrinology, Medical University of Gdansk, Poland
b
INVICTA Laboratory of Molecular Biology, Prophylactic Centre, Rajska 10, 80-850 Gdansk, Poland
c
Department of Pathology, Maritime Hospital, Gdynia, Poland
d
Department of Oncology and Radiotherapy, Medical University of Gdansk, Poland
e
Laboratory of Cancer Research and Clinical Oncology, Department of Medical Oncology, University of Antwerp (UA/UZA), Wilrijk, Belgium
Received 24 April 2006
Available online 6 December 2006

Abstract

Objective. High-risk types of HPV are etiological factors in cervical cancer. Lymph node involvement in cervical cancer patients reduces
5-year survival rates by 25–60%. However, the influence on survival of HPV DNA positivity in histopathologically negative lymph nodes remains
unresolved.
Methods. The study included 116 of 148 patients who underwent Piver type III radical hysterectomy and pelvic lymphadenectomy and who
showed HPV DNA positivity in the primary lesion. Lymph node tissues were tested for the presence of HPV DNA, using a PCR technique.
Results. We found the presence of HPV DNA sequences in lymph nodes dissected intraoperatively in 81 (69.83%) cases. In analysis, we
compared patients from 3 groups: HPV− and metastatic-negative (LN HPV-M−); HPV-positive metastatic-negative (LN HPV+M−); and
metastatic-positive (LN M+). We discovered that survival in groups LN M+ and LN HPV+M− did not differ statistically (p = 0.37). However, the
survival periods in these two groups differed when compared with LN HPV-M− patients (p < 0.001). Using Cox's proportional hazards model, we
found that the presence of lymph node HPV DNA, and FIGO stage, and primary lesion volume were independent parameters correlating with
survival and mortality risk.
Conclusion. We conclude that the presence of HPV DNA in lymph nodes is an early sign of metastasis and should be treated as such in
prognostic outlook and planning the therapeutic strategy.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Cervical cancer; Lymph node; HPV; Metastasis

Introduction HPV positivity in histopathologically negative lymph nodes.

The presence of DNA of human papillomavirus (HPV DNA)
in cervical cancers varies widely, from 60–99%. It depends on
the HPV detection method, the quality of the investigated
material and the population covered [1]. Hence, the significance
of the presence of HPV DNA in the primary lesion is still under
discussion. The problem of significance also arises in cases of
⁎ Corresponding author. Fax: +48 58 763 14 76.
E-mail address: wojciech.sliwinski@invicta.pl (W. Sliwinski).
0090-8258/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygyno.2006.10.018
Lymph node status is considered to be the most important
prognostic factor after FIGO stage. It is suggested that lymph
node involvement reduces 5-year survival rates by 25 to 60%,
within stages (except IA) [2]. However, the influence of lymph
node HPV DNA positivity remains unresolved [3–8]. Nume-
rous pathological parameters affecting survival rate are also
recognized. Well established prognostic parameters could allow
definition of the most suitable treatment.
In the present prospective study, we attempted to answer the
question of whether or not lymph node positivity for HPV DNA
is of prognostic value in pathologically negative lymph nodes in
722 K. Lukaszuk et al. / Gynecologic Oncology 104 (2007) 721–726

cases of cervical cancer. If it is, the question then arises of
whether or not there are any known clinical or pathological
parameters that could predict the presence of HPV DNA in
lymph nodes, such that lymph node dissection could be avoided
or this procedure could be limited to “higher risk” patients.
Materials and methods
The study included 116 of 148 patients who underwent Piver type III radical
hysterectomy and pelvic lymphadenectomy in the Second Department of
Obstetrics and Gynecology, Medical University of Gdansk, in 1998 and 1999 [9]
and who showed HPV DNA positivity in the primary lesion. HPV DNA status
was assessed routinely before the surgical procedure. We assessed cervical
carcinoma clinical stage according to FIGO classification, using standard
techniques. Histologic examination of specimens obtained by means of
colposcopically guided biopsy, D and C procedures or cold knife cone biopsy
of the cervix aided diagnosis. Gynecological examination, cystoscopy,
rectoscopy and standard imaging procedures such as chest X-ray and
ultrasonographic examination of the abdominal cavity were also carried out.
Patients with cancer staged IIA and IIB qualified for surgical treatment carried
out by two experienced gynecological oncologists. Lymphadenectomy was
limited to obturator and external iliac lymph nodes. A positive pathological
intraoperative result in lymph node evaluation resulted in widening the surgical
procedure to the common iliac lymph nodes and, if these were positive, to the
para-aortic lymph nodes too. After the surgical procedure, 111 from 116 patients
were subjects of supplementary radiotherapy. All patients were followed-up at
the out-patient clinic of the Second Department of Obstetrics and Gynecology at
regular 3-month intervals for the first 2 years and then twice yearly. The study
was approved by Ethical Committee for Biomedical Studies Medical University
of Gdansk (TKEBN/369/97).
DNA isolation
Frozen materials from primary lesions and lymph nodes were carefully
sectioned to avoid contamination. All cancer-positive samples were chosen by
two gynecological pathology specialists. In cases of macroscopically negative
nodes, sections were apportioned for microscopic and molecular evaluation.
Slices of frozen tissue were divided one by one for histopathological examination
and HPV detection procedure. Slices were examined carefully by experienced
pathologist. Staining for cytokeratin with antibodies prepared for cytokeratin
from 40 to 67 kDa of molecular weight was performed in every case of suspicious
or unclear results in regular histopathological examination. About 25 mg
± 5.5 mg of frozen tissue was taken for DNA isolation. The samples were
incubated in 170 μl of proteinase K buffer (10 mM Tris, 1% SDS, 400 μg
proteinase K) at 50°C, overnight. DNA was extracted using silica-based
chromatography mini-columns (Genomic DNA PrepPlus; A&A Biotechnology,

Table 1
Clinical staging and pathologic data according to lymph node status
Metastasis-negative, Metastasis-negative, Metastasis-positive, Cervical cancer patients
HPV-negative (n = 35) HPV-positive (n = 20) HPV-positive (n = 61) (n = 116)
Age (mean) ± SD 51.74 ± 10.66 48.94 ± 11.28 48.83 ± 9.59 49.7 ± 10.21
FIGO stage (%)
IB 21 (60) 10 (50) 33 (54.1) 64 (55.2)
IIA 8 (22.9) 5 (25) 19 (31.1) 32 (27.6)
IIB 6 (17.1) 5 (25) 9 (14.8) 20 (17.2)
Histopathology (%)
Squamous cell carcinoma
Large cell keratinizing 9 (25.7) 7 (35) 10 (16.4) 26 (22.4)
Large cell 25 (71.4) 11 (55) 48 (78.7) 84 (72.4)
nonkeratinizing
Adenocarcinoma and 1 (2.9) 2 (10) 3 (4.9) 6 (5.2)
adenosquamous
Histopathological lymph node
metastasis (%)
− 35 (100) 20 (100) 0 55 (47.4)
+ 0 0 61 (100) 61 (52.6)
Depth of cervical invasion (%)
<10 mm 9 (25.7) 6 (30) 3 (4.9) 18 (15.5)
>10 mm 26 (74.3) 14 (70) 58 (95.1) 98 (84.5)
Volume of primary lesion (%)
<20 cm3 18 (51.4) 8 (40) 13 (21.3) 39 (33.6)
>20 cm3 17 (48.6) 12 (60) 48 (78.7) 77 (66.4)
Corpus invasion (%)
Not across internal isthmus 21 (60) 10 (50) 20 (32.8) 51 (43.9)
Across internal isthmus 14 (40) 10 (50) 41 (67.2) 65 (56.1)
Vaginal invasion (%)
− 27 (77.1) 11 (55) 32 (52.5) 70 (60.3)
+ 8 (22.9) 9 (45) 29 (47.5) 46 (39.7)
Parametrial invasion (%)
− 33 (94.3) 16 (80) 52 (85.2) 101 (87.1)
+ 2 (5.7) 4 (20) 9 (14.8) 15 (12.9)
Type of HPV in primary
lesion (%)
HPV16 32 (91.4) 20 (100) 58 (95.2) 110 (94.8)
HPV18 0 0 1 (1.6) 1 (0.9)
HPV31 2 (5.7) 0 1 (1.6) 3 (2.6)
HPV33 1 (2.9) 0 1 (1.6) 2 (1.7)
 K. Lukaszuk et al. / Gynecologic Oncology 104 (2007) 721–726 723

Gdansk, Poland), according to the manufacturer's recommendations. DNA was negative (LN HPV-M−); HPV-positive, metastatic-negative
stored at − 30 °C. The PCR procedure for each sample was performed twice, by (LN HPV+M−); and metastatic-positive (LN M+). Difference
different researchers, with an interval of more than two weeks, starting with
repeated DNA isolation. Only concordant results allowed inclusion of the
in survival between LN HPV-M− patients and LN HPV+M−
material for further evaluation. did not differ statistically (p = 0.06). We also discovered that
survival in patient groups LN M+ and LN HPV+M− did not
HPV DNA detection differ statistically (p = 0.37). However, the survival periods in
these groups differed when compared with that in LN HPV-M−
PCR was performed for separate parallel amplification of E6 (pU-1M/pU- patients (p < 0.001). (Fig. 2).
2R primer pair) [10] and L1 (MY09/MY11 primer pair) [11] HPV genes. The
Because we found a strong correlation between lymph node
PCR reactions were carried out in 25 μl of a reaction mixture containing
10 mM Tris–HCl (pH 8.81), 5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, HPV DNA positivity and poor prognosis, we attempted to
200 μmol each of dNTPs, 10 μmol each of the 8 primers, 0.5 μl (2 U/μl) identify clinical and/or pathological parameters that might
DyNAzyme II DNA polymerase (FinnZyme, Finland) and 60 ng (5 μl) correlate with the presence of lymph node HPV DNA. We took
template DNA. A 2-min denaturation step at 94°C was followed by 30 cycles into account the following clinical parameters: age at diagnosis,
of amplification in a PCR thermocycler (TRIO-BIOMETRA, Germany). Each
menarche and menopause, menstrual cycle length and dis-
cycle included a denaturation step at 94°C for 1 min, a primer-annealing step
at 55°C for 2 min and a chain elongation step at 72°C for 2 min. The final orders, number of pregnancies and deliveries, number of
elongation step was prolonged by 5 min to ensure complete elongation of the abortions (spontaneous and induced), clinical stage (FIGO
amplified DNA. During the detection stage, 10-μl samples of PCR products classification), histologic tumor types, depth of cervical
were subjected to electrophoresis on a 10% polyacrylamide gel and silver invasion, initial size of primary lesion, lymph node metastasis
stained: incubation for 6 min in 2% nitric acid, then kept for 30 min in a 1 mg/
status and parametrial, uterine and vaginal involvement.
ml AgNO3 solution. The bands were visualized by incubating the gel in 3%
Na2CO3. Transilluminated gel images were digitalized and analyzed by means Assessing clinical parameters in LN HPV+ and LN HPV−
of the Gel Doc 2000 Documentation System (Bio-Rad, USA). groups, we discovered no statistically significant differences.
Assessment of pathologic parameters vs. LN HPV DNA
Statistical analysis status revealed statistically significant relationships in most
cases, apart from histologic tumor type, depth of cervical
It is accepted that lymph node involvement reduces 5-year survival rates by invasion and parametrial involvement (Table 2).
25 to 60%, within stages [2]. Therefore, we planned to randomize 120 patients to
detect a 25% difference in survival probabilities between lymph node HPV
We also investigated the association between clinical and
DNA-positive and -negative groups. The significance level (alpha) was set at pathologic parameters and survival rate in both groups. We
0.05 with a power of 0.8. STATISTICA for Windows 6.0 (StatSoft, Inc., Tulsa, discovered no significant associations as regards: age at
OK, USA) was used for statistical analysis. The chi-square test for determination diagnosis (HPV+: p = 0.82, HPV−: p = 0.17), age at
of independence of variables and multiple logistic regression analysis for menarche (HPV+: p = 0.97, HPV−: p = 0.52), menopausal
predictors for lymph node metastasis were applied. Survival probabilities were
calculated by means of the Kaplan–Meier method, with differences between age (HPV+: p = 0.93, HPV−: p = 0.55), number of pregnan-
survival probabilities calculated by means of the log-rank test. Multivariate cies (HPV+: p = 0.27, HPV−: p = 0.77), number of deliveries
analyses were performed by means of the Cox regression model. The level of (HPV+: p = 0.86, HPV−: p = 0.48), number of abortions
statistical significance was set at p < 0.05. (HPV+: p = 0.36, HPV−: p = 0.25). Clinical stage according to
FIGO classification correlated with the survival of LN HPV+
Results patients (p < 0.001), in contrast to LN HPV− patients (p = 0.07). In
LN HPV+ patients, we observed 18 deaths at stage Ib, 18 at stage
The clinical staging and pathologic data are presented in IIa and 13 at stage IIb. We observed a statistically significant
Table 1.
We found the presence of HPV DNA sequences in lymph
nodes dissected intraoperatively in 81 (69.83%) cases. In analysis
we compared patients in two groups: lymph node HPV-positive
(LN HPV+) and lymph node HPV-negative (LN HPV−).
The mean postoperative follow-up period in our study was
39.2 ± 26.2 (SD) months in the LN HPV+ group and 54.8 ± 23.1
in the LN HPV− group. In the LN HPV+ group, there were 49
(60.5%) deaths and the mean postoperative survival period was
22.9 ± 16.8 (SD) months (upper quartile 29.1). In the LN HPV−
group, the figures were 9 (25.7%) deaths and 24.1 ± 16.9 months
(upper quartile 40.7), respectively (Fig. 1).
We thus confirmed a statistically different survival rate in the
two groups (p<0.001), the mortality rate being higher in the LN
HPV+ group. The mortality rate was comparable in the two
groups during the first two years of observation. Subsequently,
it was greater in the LN HPV+ group. Because most of the LN
HPV+ cases were also pathologically positive, we decided to Fig. 1. Kaplan–Meier survival curves stratified according to HPV-positive and
analyze the survival period in 3 groups—HPV- and metastatic- -negative lymph node groups.
724 K. Lukaszuk et al. / Gynecologic Oncology 104 (2007) 721–726

Table 3
Clinical and histopathological parameters in Cox's proportional hazards analysis
Analyzed data p value Relative risk
Clinical FIGO stage <0.001 2.04
Lymph node HPV DNA presence 0.04 2.31
Lymph node metastasis 0.57 1.23
Primary lesion volume 0.04 2.63
Depth of cervical invasion 0.59 0.71
Corpus invasion 0.17 1.25
Parametrial invasion 0.49 0.77
Vaginal invasion 0.94 0.98

(p = 0.33). Vaginal involvement of the primary tumor turned out

Fig. 2. Kaplan–Meier survival curves stratified according to HPV DNA and
metastatic status.
difference in survival rate between patients at stages Ib and IIa
(p = 0.01), but we did not observe such a difference between stages
IIa and IIb (p = 0.15). We noted that in LN HPV+ and LN HPV−
patients, primary lesion volume correlated with survival
(p = 0.001 and p = 0.03, respectively); it was statistically sig-
nificantly shorter in patients who developed a tumor volume
exceeding 20 cm3. We did not confirm an influence of cervical
invasion depth on survival (LN HPV+ group: p = 0.03, LN HPV−
group: p = 0.66). We discovered that parametrial involvement
statistically significantly shortened the survival period in the LN
HPV− group (p = 0.01) but not in the LN HPV+ group
not to influence survival in either group (LN HPV+ group:
p = 0.14, LN HPV− group: p = 0.38). We evaluated the
association between independent clinicopathological variables
and survival by means of Cox's proportional hazards model. In
statistical analysis, we took into account pathological para-
meters, the presence of lymph node HPV DNA and FIGO stage.
The data are presented in Table 3.
We found that FIGO stage, presence of lymph node HPV
DNA and primary lesion volume were independent parameters
correlating with survival and mortality risk in patients suffering
from cervical carcinoma.
Discussion
It is well established that lymph node involvement is the
most important factor indicating poor prognosis after FIGO
stage [2]. The problems still under evaluation are as follows: Is

Table 2
Histopathological parameters versus LN HPV DNA status
HPV-positive HPV-negative Chi-square
(n = 81) (n = 35) test
Histological type of cancer
Squamous keratinizing 17 9 p = 0.68
Squamous nonkeratinizing 59 25
Adenocarcinoma and 5 1
adenosquamous
Histopathological lymph
node metastasis
− 20 35 p < 0.001
+ 61 0
Depth of cervical invasion p = 0.04
<10 mm 9 9
>10 mm 72 26
Volume of primary lesion
<20 cm3 21 18 p = 0.007
>20 cm3 60 17
Corpus invasion
Not across internal 30 21 p = 0.02
isthmus
Across internal isthmus 51 14
Vaginal invasion
− 43 27 p = 0.01
+ 38 8
Parametrial invasion
− 68 33 p = 0.12
+ 13 2
 K. Lukaszuk et al. / Gynecologic Oncology 104 (2007) 721–726
the presence of HPV DNA essential for cancer cell transforma-
tion and are there any HPV DNA-negative cancers? The
prevalence of HPV DNA in cervical cancers varies from 40% to
almost 100% [12–16]. The reason could depend on many
factors, which have been widely discussed [8]. The main
problems are as follows: methods of HPV detection used,
quality of the investigated tissue, the amount of tumor cells in
sampled tissue and sample contamination. At present, the use
of commercial hybridization tests is limited as regards clinical
use. Most investigators use PCR techniques, being more
sensitive and much more flexible in the implementation of
new ideas. We use a modified method involving E6-detecting
PCR consensus primers, with a plasmid-prepared internal
standard [17]. The quality of the investigated tissue seems to
us to be the most important factor. After 7 years of experience
of PCR, we consider paraffin-embedded samples to be highly
questionable material. Hence, we decided to design a
prospective study, based on freshly frozen intraoperative
samples only. The lack of standardization of the amount of
cancer cells could influence the results. In our study, each
cancer-positive sample was chosen by two specialists in
gynecological pathology. In cases of macroscopically negative
nodes, sectioned material was apportioned for microscopic and
molecular evaluation. The contamination problem, in our
opinion, is crucial. We avoided false-negative results thanks to
our internal standard in PCRs. As regards false-positives, we
decided to treat each sample twice, using different researchers
and a more than two-week interval, starting with a repeated
DNA isolation procedure. Only concordant results allowed
inclusion of the material for further evaluation. Since the
question of the presence of HPV DNA in cervical cancer
tissues is still under evaluation, we decided to exclude samples
that were HPV DNA-negative in the primary lesion. This
allowed us to eliminate the influence of the cancer-positive
HPV-negative lymph node in HPV-negative cancers.
To the best of our knowledge, presented in this paper is the
largest prospective investigation based on freshly frozen
intraoperatively dissected lymph nodes. The presence of
metastases in lymph nodes is a very important prognostic
factor and it is also taken into account in cervical cancer
treatment protocols as regards adjuvant radiotherapy. Hence, the
question of lymph node HPV DNA positivity needs to be
clarified. In the material presented here, we found the presence
of metastases in lymph nodes to be a very strong prognostic
indicator of poor outcome [18]. This confirmed many results
published previously, but we also affirm the fact that metastatic-
negative HPV DNA-positive tumors reflect an equally bad
prognosis as metastatic ones. These groups showed similar
Kaplan–Meier survival curves (p = 0.37). Difference in survival
between metastatic-negative HPV DNA-negative patients and
metastatic-negative HPV DNA-positive was very close to
statistical significance (p = 0.06). Deaths in the group of patients
with metastatic-negative HPV DNA-negative occurs during
first two years of follow-up. Deaths after two years of
observation mostly concern only patients with HPV DNA-
positive lymph nodes. We still do not know the role of HPV
DNA in lymph nodes without pathological metastases. It could
725
reflect undigested DNA in immunocompetent cells, or unde-
tectable hematogenic dissemination, and it could therefore
possibly be used as a diagnostic marker to predict recurrence in
these patients [3]. Previously published studies based on large
groups of patients were performed on archival material only
[8,19]. In both cases, the presence of HPV DNA in lymph node
metastatic-negative cancers was a poor prognostic factor. In our
prospective study, and using Cox's proportional hazards model,
we found that the presence of lymph node HPV DNA, and also
FIGO stage and primary lesion volume were independent
parameters correlating with survival and mortality risk. This
strongly supports the opinion that the presence of HPV DNA in
lymph nodes is an early sign of metastasis and could predict
poor prognosis. Opposite results were stated in the study
published by Fule et al. [19]. In this paper HPV presence in
lymph node shows no correlation with survival. This paper also
contains information, that 35% of metastasis-bearing lymph
nodes, taken from patients with primary tumor positive for HPV
DNA, are free from HPV DNA. Besides, they found, in 16% of
cases HPV-positive lymph nodes associated to a virus-negative
primary tumor. In our opinion these results support statement
that PCRs performed from paraffin-embedded samples are
highly questionable rather then statement that lymph nodes
HPV status is not important for cervical cancer patients survival.
On the basis of these results, we tried to find parameters that
could predict the presence of HPV DNA in lymph nodes. We
did not find any clinical or pathological feature that could be
unambiguously recognized as a predictive factor of the presence
of lymph node HPV DNA. The strongest one was a metastatic
status of lymph nodes (p < 0.001). HPV DNA-positive lymph
nodes were also more frequent in patients with larger primary
lesion volumes (p = 0.007), corpus invasion (p = 0.02) and
vaginal invasion (p = 0.01), but not parametrial invasion
(p = 0.12). These parameters have been presumed to affect
survival independently in different studies [20–25]. We also
found no parameter that could be a predictive factor of HPV
DNA lymph node absence. This might indicate performing
nodal dissection in all cases.
We conclude that the presence of HPV DNA in lymph nodes
is probably an early sign of metastasis and should be treated as
such. We suggest widening our investigation to a multicenter
prospective study. As suggested by Pilch et al. [26],
standardization of the molecular method is, of course, essential,
and in our opinion carrying out HPV DNA detection on the
same material in different centers should be of great value.
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