2234 Vol.

9, 2234 –2240, June 2003 Clinical Cancer Research

Overexpression of Hypoxia-inducible Factor 1␣ Indicates

Diminished Response to Radiotherapy and Unfavorable
Prognosis in Patients Receiving Radical Radiotherapy
for Cervical Cancer1

Barbara Bachtiary, Monika Schindl, regression). No association between HIF-1␣ expression and
Richard Pötter, Bettina Dreier, infection with different HPV types could be found.
Conclusions: Overexpression of HIF-1␣ has predictive
Thomas Hendrik Knocke, and prognostic significance in cervical cancer patients re-
Johannes A. Hainfellner, Reinhard Horvat, and ceiving curative radiation therapy. Possibly, expression of
Peter Birner2 HIF-1␣ could serve as intrinsic marker of hypoxia in cervi-
Department of Radiotherapy and Radiobiology [B. B., R. P., B. D., cal cancer.
T. H. K.], Department of Gynecology and Obstetrics [M. S.], Institute
of Neurology [J. A. H.], and Clinical Institute of Pathology [R. H.,
P. B.], University of Vienna, A-1090 Vienna, Austria
INTRODUCTION
Angiogenesis is considered as essential for growth and
progression of solid malignant tumors (1). If this formation of
ABSTRACT new blood vessels is not sufficient to provide enough O2 to
Purpose: The purpose is to investigate the impact of proliferating tumor cells, tissue hypoxia results. However, a
hypoxia-inducible factor (HIF)-1␣ expression on response to dense network of newly formed capillaries within tumors does
radiotherapy and prognosis of patients with primary irra- not necessarily imply that these capillaries are fully functional,
diated cervical cancer. Because human papillomavirus and thus hypoxia is not present (2). Therefore, tissue hypoxia is
(HPV) oncoprotein E6 might interact with HIF-1␣ in vari- a common feature of most solid tumors, often with heterogene-
ous pathways, we also investigated the relation of HIF-1␣ ous O2 levels within different regions of the individual tumors.
and HPV status. Tissue hypoxia within malignant tumors is considered an
Experimental Design: Expression of HIF-1␣ was inves- important factor for response to treatment. Hypoxic tumor cells
tigated by immunohistochemistry in 67 specimens of pa- are resistant to radiation therapy (3), and in cervical cancer
tients who had received radical radiotherapy for cervical treated with radiotherapy, low oxygen tension assessed by po-
cancer stages IB–IIIB. HPV analysis was performed using larographic oxygen needle electrodes are associated with in-
type-specific PCR, cloning, and sequencing. Survival analy- creased rate of metastasis and poor survival (4 –7).
sis was performed using univariate and multivariate analysis. The cellular adaptation to hypoxic stress is highly complex
Results: Immunohistochemistry revealed expression of and depends on up-regulation of genes supporting anaerobic
HIF-1␣ in 72.1% of the tumor samples. In 16 (23.9%) cases, metabolism and new blood vessel recruitment. The transcription
there was a weak expression, in 25 (37.3%) a moderate factor HIF-1␣3 is a key factor in this adaptation (8, 9).
expression, and in 7 cases (10.4%) a strong expression of We have recently demonstrated that HIF-1␣ is closely
HIF-1␣. Nineteen samples (28.4%) were considered negative associated with dismal prognosis in early-stage cervical cancer
for HIF-1␣ expression. Strong/moderate expression of treated by primary surgery (10).
HIF-1␣ was associated with only partial response to radio- Because HIF-1␣ has been recently shown to be associated
therapy (P ⴝ 0.037, ␹2 test). Strong/moderate expression of with response to radiotherapy in oropharyngeal cancer (11),
HIF-1␣ was also an independent prognostic factor for head and neck cancer (12), in early esophageal cancer (13), and
shorter progression-free survival (P ⴝ 0.036, Cox regres- in nasopharyngeal carcinomas (14), the aim of this study was to
sion) and cervical cancer-specific survival (P ⴝ 0.04, Cox investigate the impact of HIF-1␣ expression on response to
radiotherapy and prognosis of patients with primary irradiated
cervical cancer. This is of particular interest because extensive
tissue hypoxia is considered as a main cause for treatment
failure in radio-oncology (15).
Received 5/6/02; revised 12/3/02; accepted 1/6/03. A strong causal relationship between infection with HPV
The costs of publication of this article were defrayed in part by the and cervical cancer has been established (16), and the viral
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
This study was supported by Jubiläumsfonds der Oesterreichischen
Nationalbank Grant 8666.
2 3
To whom requests for reprints should be addressed, at University of The abbreviations used are: HIF-1␣, hypoxia-inducible factor 1␣;
Vienna, Institute of Clinical Pathology, Währinger Gürtel 18-20, HPV, human papillomavirus; FIGO, International Federation of Gyne-
A-1090 Vienna, Austria. Phone: 43-1-40400-3650; Fax: 43-1-4053402; cology and Obstetrics; PFS, progression-free survival; CCSS, cervical
E-mail: [email protected]. cancer-specific survival.

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 Clinical Cancer Research 2235

oncoproteins E6 of various HPV types differ in oncogenic the staining intensity (11, 22). The percentage of positive cells
potential (14). Because E6 might interact with HIF-1␣ in vari- was rated as follows (11, 22): cases with ⱕ10% positive cells
ous pathways (17, 18), we also investigated the relation of were rated as negative (no points attributed), regardless of
HIF-1␣ expression and HPV status in our collective of patients. staining intensity; 2 points, 11–50% positive cells; 3 points,
51– 80% positive; and 4 points, ⬎80% positive cells. The stain-
ing intensity was rated as follows: 1 point, weak intensity; 2
MATERIALS ANDMETHODS points, moderate intensity; and 3 points, strong intensity. Points
Patients. Formalin-fixed, paraffin-embedded biopsy for percentage of positive cells and staining intensity were
samples of 67 patients with primary cervical cancer FIGO stage added, and specimens were attributed to four groups according
IB–IIIB were included in the study. All patients underwent to their overall score: negative, ⱕ10% of cells stained positive,
primary radiotherapy at the Department of Radiotherapy and regardless of intensity; weak expression, 3 points; moderate
Radiobiology, University Hospital of Vienna, between Novem- expression, 4 –5 points; and strong expression, 6 –7 points. Two
ber 1993 and October 2001. independent investigators blinded to clinical data performed the
Bulky disease was defined as (a) visible cervical tumor analysis. Specimens scored differently by the two investigators
with the largest diameter ⬎4 cm or (b) a cervix expanded to ⬎4 were reinvestigated together using a multiheaded microscope. In
cm as a result of tumor invasion. Computerized topography of addition, the presence of necrotic areas within tumor formations
the pelvis and abdomen was performed to determine the nodal and HIF-1␣ expression in tumor cells directly adjacent to these
status. Patients with enlarged para-aortic and/or common iliac areas was evaluated.
nodal involvement were considered lymph node positive HPV Analysis. DNA was extracted from 10-␮m thick
All patients underwent primary radiotherapy with the in- paraffin-embedded tissue sections of 64 cases for HPV-analysis
tention of achieving cure. The treatment consisted of external as described previously (23, 24). In 3 cases, not enough material
beam radiotherapy with a four-field box technique to the pelvis was left for analysis. All samples were tested for ␤-globin (25),
and to the para-aortic region (depending on the stage), consist- which served as internal control for the integrity of the extracted
ing of a total dose of 40 –50 Gy (median, 48.6 Gy) applied in template, followed by consensus HPV-PCR (GP5⫹/GP6⫹; Ref.
daily fractions of 1.6 –2 Gy. External beam irradiation was 26) from the L1 region.
carried out using a 25-MV linear accelerator, with central Typing of HPV DNA was performed by type-specific
shielding in anterior-posterior portals, depending on the stage of PCR: HPV-16 (E7/E1: 698 –917); HPV-18 (E6/E7: 533–705;
disease and tumor volume. Three to six fractions of intracavitary Ref. 27); HPV-31 (E5: 3835–3989; Ref. 27); HPV35 (E7:
high dose-rate brachytherapy were applied in weekly fractions 610 – 840; Ref. 27); HPV-33 (E6: 265–396); and HPV-45 (E6/
of 7 Gy each to point “A,” depending on the tumor stage and E7: 548 – 694).
tumor volume (19). After PCR amplification, the PCR products were separated
The follow-up investigations were commenced 4 weeks on a 2% Tris-acetate-EDTA-agarose gel, followed by visualiza-
after the last treatment and were performed thereafter at tion of the DNA fragments under UV light.
3-month intervals. Response to treatment was evaluated by To detect infection with other HPV types, consensus PCR
clinical examination and by appropriate imaging studies (in all products were cut out and purified using the QIAquick Gel
cases computerized topography, whenever appropriate, mag- Extraction kit (Qiagen, Hilden, Germany) according to the man-
netic resonance imaging and ultrasound) at 3 months. Persistent ufacturer’s instructions. The purified consensus HPV DNA was
disease was defined as disease within the pelvis 3 months after cloned into pTargeTTM Mammalian Expression Vector System
completion of radiotherapy. Recurrent disease was defined as (Promega, Madison, WI), followed by thermocycle sequencing
local (within pelvis) or distant (outside the pelvis). (Amersham Life Science, Piscataway, Ohio).
Immunhistochemistry. For immunohistochemical de- As additional positive control, we investigated HPV status
tection of HIF-1␣, a 4-␮m tissue section was deparaffinized in in selected cases with signal-amplified in situ hybridization with
xylene followed by microwave treatment in for 30 min in 0.01 probes against HPV-16, HPV-18, HPV-31, and HPV-33 as
M citrate buffer (pH 6.0) at 600 W. After cooling for 20 min and described previously (28).
washing in PBS, endogenous peroxidase was blocked with Statistical Methods. For statistical analysis, two groups
methanol containing 0.3% hydrogen peroxide for 30 min, fol- of patients were formed with regard to HIF-1␣ expression: i.e.,
lowed by incubation with PBS containing 10% normal goat absent or low expression and strong or moderate expression.
serum for 30 min. Specimens were incubated overnight at ⫹4°C Association between HIF-1 ␣ expression and clinicopath-
with a monoclonal anti-HIF-1␣ antibody (no. H72320; BD ological factors were analyzed by using Mann-Whitney test and
Transduction Laboratories, Franklin Lakes, NJ; Refs. 20, 21) at the ␹2 test.
a dilution of 1:25. Detection of immunostaining was performed PFS was defined as the period from end of therapy to the
using the ChemMate kit (Dako, Glostrup, Denmark) and 3,3⬘- date of the first documented evidence of recurrent disease.
diaminobenzidine as chromogene. For positive control, HIF-1␣ CCSS was calculated from the date of diagnosis to death;
immunostaining was also performed on two samples of ovarian patients who survived until the end of the observation period
cancer with known strong expression, which have also been were censored at their last follow-up visit. Patients who died
used in a previous study (22). For negative control, the primary because of other causes than cervical cancer were censored at
antibody was replaced by nonimmune isotypic antibodies. their date of death. Survival curves were calculated using
Nuclear expression of HIF-1␣ was determined by assessing Kaplan-Meier estimates, and differences between groups were
semiquantitatively the percentage of decorated tumor cells and tested by log-rank test. Multivariate survival analysis was per-

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2236 HIF-1␣ in Cervical Cancer Patients and Radiotherapy

Table 1 Patient characteristics and HIF-1␣ expression

Patients HIF-1␣ expression
Strong/moderate Absent/weak
n % n % n % P
FIGO stage 0.83
IB 6 8.9 3 50 3 50
IIa 34 50.8 15 44.1 19 55.9
IIIb 27 40.3 14 51.9 13 48.1
Tumor size 0.56
⬍4 cm 19 28.4 8 42.1 11 57.9
⬎4 cm 48 71.6 24 50 24 50
Histology 0.38
Squamous 59 88.1 27 45.8 32 54.2
Adenocarcinoma 8 11.9 5 62.5 3 37.5
Necrosis ⬍0.001
With necrosis 29 43.3 22 75.9 7 24.1
Without necrosis 38 56.7 10 26.3 28 73.7
Lymph nodes 0.30
Negative 46 68.7 20 43.5 26 56.5
Positive 21 31.3 12 57.1 9 42.9
Grading 0.59
Grade 1 7 12.1 2 28.6 5 71.4
Grade 2 34 58.6 17 50 17 50
Grade 3 17 29.3 8 47.1 9 52.9
HPV status 0.29
Negative 6 8.9 2 33.3 4 66.7
Single HPV-16 20 29.9 9 45 11 55
Other single HPV types 10 14.9 4 40 6 60
Multiple HPV-16 ⫹ HPV-33 16 23.9 9 60 6 40
Other multiple HPV types 12 17.9 3 50 3 50
HPV positive/subtype unknown 3 4.5 3 100 0 0
a
IIA, n ⫽ 3; IIB, n ⫽ 31.
b
IIIA, n ⫽ 9; IIIB, n ⫽ 18.

formed according to the Cox proportional hazards model.

HIF-1␣ expression (absent/low versus moderate/strong), tumor
size (bulky versus nonbulky), patients’ age, nodal status, FIGO
stage, and histological grading were included in the regression
model.
For all statistical tests, P ⱕ 0.05 was considered significant.

RESULTS
Clinical and histopathological patient characteristics are
given in Table 1. Fifty-five patients had a complete clinical
response to radiotherapy, whereas 12 patients (17.9%) had in-
complete response (n ⫽ 7) or lymphogene disease progression
(n ⫽ 5) after radiation therapy. Median follow-up time was 27
months (range, 5– 84 months). During this observation period,
23 patients experienced both local and/or distant relapse, and 35
patients (all patients with incomplete response to radiotherapy
and all patients with recurrent disease) died because of cervical
cancer. Fig. 1 A specimen of cervical cancer with strong expression of HIF-
HIF-1␣ Expression. Immunhistochemistry revealed 1␣: note the distinct nuclear signal. Immunoperoxidase, original mag-
nification, ⫻200.
decoration by the HIF-1␣ antibody in 71.6% of the tumor
samples. In 16 (23.9%) cases, there was a weak expression, in
25 (37.3%) a moderate expression, and in 7 cases (10.4%) a
strong expression of HIF-1␣ (Fig. 1). Nineteen samples (28.4%) HIF-1␣ expression. Tumors with necrotic areas had signifi-
were considered as negative for HIF-1␣ expression. cantly more often strong/moderate HIF-1␣ expression compared
Twenty-nine tumors (43.3%) were found to have necrotic with the other cases (median expression: moderate versus weak,
areas, and 27 of these tumors (93.1%) showed perinecrotic P ⬍ 0.001, ␹2 test; Table 1). No significant association between

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 Clinical Cancer Research 2237

was found in 2 patients as a single HPV type (8.3%) and in 22

additional patients as part of multiple HPV types (91.7%). The
most commonly found combination of multiple HPV types
involved HPV-16 plus HPV-33 (n ⫽ 15). Other high-risk types
(single and/or multiple HPV types) included HPV-18 (7 pa-
tients), HPV-31 (8 patients), HPV-45 (4 patients), and HPV-58
(1 patients) and the low-risk types HPV-73 (2 patients) and
HPV-69 (1 patient).
For analyzing the influence of various HPV-types on
HIF-1␣ expression, HPV types were grouped as follows: no
HPV infection, n ⫽ 6 (10.3%); single HPV-16, n ⫽ 20 (34.5%);
other single HPV infection, n ⫽ 10 (17.2%); HPV-16 ⫹ HPV-
33, n ⫽ 16 (27.6%); and other multiple HPV infection, n ⫽ 12
(20.7%). No association of HIF-1␣ expression and infection
with various HPV subtypes was observed (P ⫽ 0.29, Mann-
Whitney test).
Association of HIF-1␣ Expression with Response to
Therapy. Seventy-five percent (n ⫽ 9) of the 12 patients who
had an incomplete response to radiotherapy were found to have
a strong/moderate HIF-1␣ expression before commencing ra-
diotherapy, whereas in patients with complete response to ra-
diotherapy (n ⫽ 55), strong/moderate HIF-1␣ expression was
found in only 41.8% (n ⫽ 23) of cases (P ⫽ 0.037; ␹2 test).
Survival Analysis. When survival of patients with
strong/moderate expression of HIF-1␣ was compared with sur-
vival of patients with absent/weak expression of HIF-1␣,
Kaplan-Meier analysis (log-rank test) revealed a significant
influence of HIF-1␣ expression on PFS (P ⫽ 0.011; Fig. 2A)
and CCSS (P ⫽ 0.006; Fig. 2B). Other factors associated with
shortened PFS and CCSS in univariate analysis were tumor
size ⬎ 4 cm (P ⫽ 0.005 and P ⫽ 0.004, respectively), positive
lymph nodes (P ⫽ ⬍ 0.001 and P ⫽ ⬍ 0.002, respectively), and
a more advanced FIGO stage (P ⫽ 0.039 and P ⫽ 0.034,
respectively; Table 2).
The 3-year CCSS rate was 71% in patients with absent/
weak expression of HIF-1␣ (median CCSS time, 62 months),
whereas in patients with strong/moderate HIF-1␣ expression, it
was 29% (median CCSS time, 24 months; Table 2).
Fig. 2 A, PFS in 67 patients with cervical cancer stage I–III with
strong/moderate expression of HIF-1␣ (a) and absent/weak expression The 3-year PFS rate was 53% in patients with absent/weak
of HIF-1␣ (b). B, cervical cancer-specific survival in 68 patients with expression of HIF-1␣ (median PFS time, 60 months), whereas
cervical cancer stage I–III with strong/moderate expression of HIF-1␣ in patients with strong/moderate HIF-1␣ expression, it was only
(a) and absent/low expression of HIF-1␣ (b). 34% (median PFS time, 13 months; Table 2).
Expression of HIF-1␣ was the only independent prognostic
factor for PFS (P ⫽ 0.049) and CCSS (P ⫽ 0.02) in multivariate
analysis. To improve the power of the analysis, lymph node
HIF-1␣ expression and FIGO stage (P ⫽ 0.664), tumor size status and tumor size were combined as follows: node negative/
(P ⫽ 0.563), histology (P ⫽ 0.377), lymphatic node involve- tumor size ⱕ 4 cm, n ⫽ 17 (25.4%); node positive/tumor size ⬎
ment (P ⫽ 0.303), and histological grading (P ⫽ 0.619) was 4 cm, n ⫽ 2 (3%); node negative/tumor size ⱕ 4 cm, n ⫽ 29
found (Table 1). (43.2%); and node positive/tumor size ⬎ 4 cm, n ⫽ 19 (28.4%).
Association of HPV Infection and HIF-1␣ Expression. At multivariate analysis of survival using this combined vari-
HPV DNA was detected in 58 of 64 (91.6%) of the patients. able, expression of HIF-1␣ and the presence of positive node
Thirty of these patients (51.7%) had tumors with one HPV DNA positives combined with tumor size ⬎ 4 cm independently
genotype, and 28 patients (48.3%) had tumors with two or more predicted outcome, as shown in Table 3.
HPV DNA genotypes.
HPV-16 was the most commonly found genotype and was
detected in 42 cases of HPV-positive tumors. HPV-16 was DISCUSSION
found in 20 patients as the only HPV type (47.6%) and in 22 Up to now only a few data exist on the expression of
patients’ cases as part of multiple HPV infection (52.4%). HIF-1␣ in cervical carcinoma. In an earlier study, we have
HPV-33 was the second most common genotype (n ⫽ 24) and shown that in patients who underwent radical surgery for early-

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2238 HIF-1␣ in Cervical Cancer Patients and Radiotherapy

Table 2 Univariate (log-rank test) analysis of survival in 67 patients with cervical cancer
PFS at 3 years Log rank CCSS at 3 years Log rank
(%) P (%) P
HIF-1␣ 0.011 0.009
Absent/weak 53 71
Strong/moderate 34 29
Tumor size 0.005 0.004
ⱕ4 cm 70 78
⬎4 cm 31 40
Nodal status ⬍0.001 0.002
Negative 55 61
Positive 15 30
Nodal status/tumor size ⬍0.001 0.004
Node negative/tumor size ⱕ 4 cm 74 65
Node positive/tumor size ⬎ 4 cm 50 50
Node negative/tumor size ⱕ 4 cm 41 20
Node positive/tumor size ⬎ 4 cm 14 9
FIGO stage 0.039 0.034
Stage IB 82 82
Stage IIA ⫹ IIB 49 55
Stage IIIA ⫹ IIIB 26 39
Histological grading 0.138 0.092
Grade 1 86 85
Grade 2 40 51
Grade 3 38 41

Table 3 Multivariate analysis of PFS and CCSS in 67 patients with them to survive and even proliferate within a hypoxic environ-
cervical cancer stage I–III ment (30). These processes contribute to the malignant pheno-
95% confidence type and to aggressive tumor behavior (31).
Relative risk interval P Radiotherapy is a major treatment modality for advanced
PFS cervical carcinomas and requires free radicals from oxygen to
HIF-1␣ 2.1 1.05 –4.2 0.036 destroy target cells, and cells in hypoxic areas were found to be
Node status/tumor size 1.9 1.15–3.13 0.012
FIGO stage 0.9 0.44 –2.1 0.9 resistant to radiation-induced cell death (2, 32). In patients with
CCSS cervical cancer, hypoxia, measured by the use of the Eppendorf
HIF-1␣ 2.1 1.03–4.19 0.04 probe, has been associated with an increased risk of relapse and
Node status/tumor size 1.8 1.12–2.89 0.02 death (4 –7). Use of the Eppendorf probe as a measure of tumor
FIGO stage 0.9 0.41–1.92 0.75
hypoxia is somewhat cumbersome and expensive. An alterna-
tive strategy for the measurements of hypoxia is to use changes
in expression of oxygen-regulated proteins.
For this purpose, the transcription factor HIF-1␣ is an
stage cervical cancer increased expression of HIF-1␣ is a strong
eligible candidate. Although HIF-1␣ expression might also be
prognostic marker (10). An association of HIF-1␣ expression
induced by oncogenic, not hypoxia-induced stimuli, tissue hy-
with response to radiotherapy was reported recently for oropha-
ryngeal cancer (11), head and neck cancer (12), early esopha- poxia is considered as the main inducer of HIF-1␣ expression in
geal cancer (13), and nasopharyngeal carcinomas (14). human tumors (33). This is in good concordance to our findings
The present results indicate for the first time a strong that HIF-1␣ expression was associated with the presence of
association between expression of HIF-1␣ and response to ra- necrotic areas.
diotherapy in cervical cancer patients. In a previous study, HIF-1␣ is a key transcription factor that was recently
Haugland et al. (29) observed a trend to worse prognosis in demonstrated to be a useful intrinsic marker for hypoxia in
patients who underwent radical radiotherapy for advanced cer- cervical cancer xenografts (34). Possibly, expression of HIF-1␣
vical cancer with strong HIF-1 expression, which did not reach might also serve as intrinsic marker of hypoxia in cervical
significance. Interestingly, Haugland et al. (29) observed a cancer tissue samples.
considerably lower rate of HIF-1␣-positive cells (⬃11%) com- HIF-1␣ activates the expression of numerous hypoxia-
pared with our study using another monoclonal antibody. response genes such as the vascular endothelial growth factor,
Hypoxia is an important factor in many pathological pro- which promotes angiogenesis, glucose transporter 1, which ac-
cesses, including tumor formation, where it has been associated tivates glucose transport, lactate dehydrogenase, which is in-
with resistance to radiotherapy, malignant progression, and me- volved in the glycolytic pathway, and erythropoietin, which
tastasis (2–7). Tumors become hypoxic because new blood induces erythropoiesis. HIF-1␣ also activates transcription of
vessels they develop are aberrant and have poor blood flow (2). nitric oxide synthase, which promotes angiogenesis and vaso-
Although hypoxia is toxic to both cancer and normal cells, dilatation (35–37).
cancer cells undergo genetic and adaptive changes that allow On the other hand, HIF-1␣ is also a potent activator of the

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 Clinical Cancer Research 2239

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Overexpression of Hypoxia-inducible Factor 1α Indicates
Diminished Response to Radiotherapy and Unfavorable
Prognosis in Patients Receiving Radical Radiotherapy for
Cervical Cancer
Barbara Bachtiary, Monika Schindl, Richard Pötter, et al.

Clin Cancer Res 2003;9:2234-2240.

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