[CANCER RESEARCH 61, 2911–2916, April 1, 2001]
Expression of Hypoxia-inducible Factor-1␣: A Novel Predictive and Prognostic
Parameter in the Radiotherapy of Oropharyngeal Cancer1
Daniel M. Aebersold, Philipp Burri, Karl T. Beer, Jean Laissue, Valentin Djonov, Richard H. Greiner, and
Gregg L. Semenza2
Department of Radiation Oncology [D. M. A., P. B., K. T. B., R. H. G.], Institute of Anatomy [V. D.], Institute of Pathology [J. L.], University of Bern, Bern, Switzerland, and
Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914 [G. L. S.]
ABSTRACT
Hypoxia has long been recognized as detrimental to the successful
treatment of malignant tumors with ionizing radiation. Because hypoxia-
inducible factor (HIF)-1␣ plays an essential role in oxygen homeostasis in
vitro, we explored the predictive potential of this factor in a cohort of 98
patients with squamous cell cancer of the oropharynx, who were treated
by curative radiation therapy. Ninety-four % of the primary tumors
showed overexpression of HIF-1␣, relative to the surrounding tissue, as
determined by immunohistochemistry. The degree of HIF-1␣ immunore-
activity correlated inversely with both the rate of complete remission of
the primary tumor (odds ratio, 0.33; P ⴝ 0.03) and lymph node metastases
(odds ratio, 0.34; P ⴝ 0.02) as well as with local failure-free survival (risk
ratio, 2.15; P ⴝ 0.006), disease-free survival (risk ratio, 2.01; P ⴝ 0.008),
and overall survival (risk ratio, 2.17; P ⴝ 0.002). The multivariate analysis
revealed the predictive power of HIF-1␣ to be independent of other
covariables. We conclude that HIF-1␣ is overexpressed in the vast ma-
jority of patients with squamous cell cancer of the oropharynx and that
the degree of expression has predictive and prognostic significance in
individuals undergoing curative radiation therapy.
INTRODUCTION
Hypoxia is a common feature of many malignant neoplasms and
exacerbates the disease in two main ways: it promotes both local and
systemic tumor progression (1) and may compromise the beneficial
effects of chemotherapeutic drugs (2) and ionizing radiation. The
deleterious effects of hypoxia on radiotherapy outcome have long
been established, based upon a formidable body of experimental
evidence. That cells maintained under anaerobic conditions are rela-
tively insensitive to radiation was first demonstrated in the 1930s (3).
This pioneering work laid the foundation for a large number of
subsequent experimental studies in vivo, which have shown unequiv-
ocally that hypoxia interferes with the response of tumors to radiation
(4). Various methodological approaches have been developed to
measure tumor hypoxia, including exogenous markers for magnetic
resonance spectroscopy and immunohistochemical staining as well as
polarographic oxygen electrodes (5). Indeed, using this latter tech-
nique, pretreatment oxygenation levels have been found to be predic-
tive of the radiation response and survival of patients with cancer of
the uterine cervix (6, 7) and of the head and neck (8, 9). Widespread
application of the technique is precluded by the invasiveness of the
method, its use being restricted to superficially located tumors.
HIF3-1 plays a pivotal role in essential adaptive responses to
hypoxia, its expression and transcriptional activity increasing expo-
nentially with decreases in levels of cellular oxygen (10 –12). Several
Received 11/15/00; accepted 1/30/01.
The costs of publication of this article were defrayed in part by the 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
Supported by the Bernese Cancer League and by a grant from the Children’s Brain
Tumor Foundation.
2
To whom requests for reprints should be addressed, at Johns Hopkins Hospital, Room
CMSC-1004, 600 North Wolfe Street, Baltimore, MD 21287-3914. Fax: (410) 955-0484;
E-mail: gsemenza@jhmi.edu.
3
The abbreviations used are: HIF, hypoxia-inducible factor; OR, odds ratio; RR, risk
ratio; CR, complete response; IMD, intratumoral microvessel density.
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dozen target genes are known to be transactivated by HIF-1, including
those encoding erythropoietin, glucose transporters, glycolytic en-
zymes, and vascular endothelial growth factor (13). HIF-1 is a het-
erodimeric basic-helix-loop-helix-PER-ARNT-SIM transcription fac-
tor that is composed of two subunits, HIF-1␣ and HIF-1␤ (10, 14).
Whereas HIF-1␤ can dimerize with several basic-helix-loop-helix-
PER-ARNT-SIM transcription factors, HIF-1␣ is unique to HIF-1; the
levels of its expression are the primary determinant of HIF-1 DNA
binding and transcriptional activity (11, 15). Induction of HIF-1␣ thus
appears to be a critical step in the hypoxic response and occurs via
increased mRNA expression, protein stabilization, nuclear localiza-
tion, and augmented activity of its transcriptional activation domains
(13). Nuclear accumulation of the protein can be detected immuno-
histochemically and has been shown to occur not only in human
malignancies and their metastases (16 –19) but also in ischemic myo-
cardial tissue (20). The present study reveals HIF-1␣ to be expressed
in the vast majority of patients with squamous cell cancer of the
oropharynx. In the same population of individuals, the local cure rate
upon radiation therapy has already been shown to depend inversely on
the IMD, a marker for angiogenic activity (21). We now show that
elevated levels of HIF-1␣ are closely and independently correlated
with reduced rates of local failure-free, disease-free, and overall
survival, thereby indicating radioresistant and aggressive disease.
PATIENTS AND METHODS
Patients. For inclusion in this study, patients were required to have pre-
sented at the Department of Radiation Oncology, Inselspital Bern, between
October 1991 and December 1997 with histologically proven squamous cell
cancer of the oropharynx and to have been treated by radical external beam
radiotherapy. A review of the radiation records identified 139 patients satis-
fying entry criteria. Forty-one of these were subsequently excluded because of
the small size of the biopsy (18 patients), previous or synchronous malignan-
cies (12 patients), irradiation after neck dissection (7 patients), intercurrent
death (2 patients), lack of follow-up (1 patient), or the existence of distant
metastasis at the onset of treatment (1 patient). The principal clinical charac-
teristics of the patient cohort are summarized in Table 1. With the approval of
the regional board of the Medical Ethics Commission, paraffin-embedded
tissue samples were obtained from the archives of the Pathology Department.
The histopathological diagnosis and grading of biopsies were reviewed by an
experienced pathologist (J. L.). Clinicopathological covariables were derived
from patient charts.
After the placement of a thermoplast mask, all patients underwent planning
computer tomography, as well as two- or three-dimensional based planning
and control using simulator and portal vision imaging devices, prior to radio-
therapy. Megavoltage radiation was delivered by means of a linear accelerator
in daily fractions, five times/week for 5– 8 weeks. A median total dose of 74
Gy (range, 54 – 80.5) was administered; 97% of the patients received at least 66
Gy. Twenty-seven patients were subjected to concomitant cisplatin-based
chemotherapy, 12 patients underwent cisplatin monotherapy, and 13 were
treated with both cisplatin and 5-fluorouracil; 1 individual received methotrex-
ate, and 1 patient received carboplatin. In patients with CR of the primary
tumor and with clinical or radiological evidence of persistent lymph node
metastasis, a neck dissection was performed.
Baseline studies included physical examination, chest X-rays, panendos-
copy of the upper aerodigestive tract, and magnetic resonance imaging or
 HYPOXIA-INDUCIBLE FACTOR-1␣ IN RADIOTHERAPY

Table 1 Bivariate correlations with HIF-1␣

HIF-1␣ staining

Frequency Weak Strong

No. of patients % of patients No. of patients % of patients No. of patients % of patients P rho/ORa
Total 98 100 56 57 36 37
Age, yr (median, 57)
⬍57 48 49 32 67 13 27 0.17 0.10
ⱖ57 50 51 24 48 23 46
T stage
1 4 4 2 50 1 25 0.56 0.06
2 8 8 5 63 3 38
3 30 31 19 63 10 33
4 56 57 30 54 22 39
N stage
0 33 34 18 55 14 42 0.52 ⫺0.07
1 13 13 7 54 4 31
2 42 43 24 57 15 36
3 10 10 7 70 3 30
Grade
Well-differentiated 11 11 5 46 6 55 0.01 ⫺0.30
Moderately 61 62 33 54 25 41
Poorly differentiated 26 27 18 69 5 19
Site
Tonsillar fossa 38 39 24 63 11 29 0.31 NA
Base of tongue 29 30 15 52 12 41
Faucial arch 21 21 12 57 9 43
Lateral/posterior wall 6 6 2 33 4 67
Vallecula epiglottica 4 4 3 75 0 0
IMD
0–80 13 13 9 69 2 15 0.48 0.07
81–110 45 46 22 49 20 44
111–130 21 21 13 62 8 38
⬎130 19 19 12 63 6 32
Smoking habits
Nonsmoker 12 12 7 58 3 25 0.22 NA
Smoker 86 88 49 57 33 38
Chemotherapy
Yes 27 28 17 63 8 30 0.66 NA
No 71 72 39 55 28 39
CR
Primary tumor
Yes 79 81 50 63 24 30 0.03 0.33
No 19 19 6 32 12 63
Lymph nodes
Yes 33 51 22 67 7 21 0.02 0.34
No 32 49 16 50 15 47
Distant metastasis
Yes 14 14 6 43 8 57 0.07 2.71
No 84 86 50 60 28 33
a
Spearman’s correlation coefficient (rho) was calculated for ordinal parameters; the OR was determined for complete remission and distant metastasis (logistic regression). NA,
not applicable.

computed tomography of the neck. During treatment, patients were examined with nonimmune serum or with the antibody diluent (Dako) served as negative
on a weekly basis. The response to treatment was assessed 2– 4 weeks after the controls.
end of therapy; the primary tumor and lymph node metastases were evaluated Tumor cell immunoreactivity was scored according to the nuclear staining.
separately. After treatment, all patients underwent clinical examination and Both the extent of staining (relative number of HIF-1␣-positive cells) and the
imaging on a regular basis. Fifty-five patients (56%) were observed until their intensity of the reaction were taken into account: ⫺, not detected; ⫹, ⬍1%
death; the median follow-up period for survivors was 2.6 years. positive cells; ⫹⫹, 1–10% positive cells with slight to moderate staining or
Immunohistochemistry. From the 98 patients, 148 paraffin-embedded bi- 10 –50% positive cells with slight staining; ⫹⫹⫹, 10 –50% positive cells with
opsies were processed for immunohistochemistry. Two or more biopsies were moderate to marked staining; ⫹⫹⫹⫹, ⬎50% positive cells with moderate to
available in 20 patients. Sections (3-␮m thick) were transferred to gelatinized marked staining. For all statistical tests, the five grades of staining were
microslides and air-dried overnight at 37°C. They were dewaxed in xylene
reduced to three: 0, not detected; I, weak (⫹/⫹⫹); II, strong (⫹⫹⫹/⫹⫹⫹⫹).
(three changes), rehydrated in a graded series of decreasing ethanol concen-
When more than one biopsy/patient was available, the highest score was
tration, and rinsed then in Tris-buffered saline [50 mM Tris/HCl (pH 7.4)
selected for further evaluation. Assessment was performed in a blinded fashion
containing 100 mM sodium chloride]. Immunostaining was performed accord-
and independently by two investigators (D. M. A., P. B.). Conflicting scores
ing to the Catalyzed Signal Amplification System (Dako, Carpinteria, CA),
which uses a streptavidin-biotin-horseradish peroxidase complex. The slides were resolved at a discussion microscope.
were initially immersed in target retrieval solution (Dako) at 97°C for 15 min Statistics. Bivariate association between ordinal variables was assessed
and thereafter treated in accordance with the manufacturer’s instructions. They using Spearman’s correlation (exact version), which yielded the correlation
were exposed to a monoclonal antibody against HIF-1␣ (H1␣67, used at a coefficient, rho. For categorical data, Pearson’s ␹ test was used. All tests of
2

dilution of 1:10,000; Ref. 17) for 30 min. The biotinyl tyramide amplification statistical significance were two-sided.
reagent was diluted 1:10 in protein blocking solution (Dako). The reaction Correlations between variables and the response to treatment as well as
product was visualized by exposing sections to 3,3-diaminobenzidine for 1 distant metastasis (time-independent outcome analysis) were determined using
min. Nuclei were lightly counterstained with hematoxylin. Sections were then the logistic regression method, implemented in both the univariate and multi-
mounted in Aquatex (Merck, Darmstadt, Germany). Tissue samples incubated variate fashion and including a backward elimination procedure to remove
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 HYPOXIA-INDUCIBLE FACTOR-1␣ IN RADIOTHERAPY
variables with P ⱖ 0.1. The qualifying criteria for inclusion in the multivariate
analysis were P ⬍ 0.1, or OR ⬍0.5 or ⬎2, in the univariate analysis.
Variables were correlated with local failure-free survival, disease-free sur-
vival, and overall survival. The analysis of local failure-free survival consid-
ered both local tumor progression after incomplete remission and local relapse
after CR as adverse events, whereas that of disease-free survival took both
local failure and distant metastasis into account. The analysis of overall
survival included death from any cause. Survival was measured from the time
when therapy was initiated to that when the first adverse event was detected or
to the date of the last follow-up. Deaths attributable to nontumor-related causes
were censored, except in the analysis of overall survival. Survival curves were
plotted according to the Kaplan-Meier method; the log rank test was used to
determine significant differences between these. A Cox regression was per-
formed to calculate the RRs. Qualifying criteria for inclusion in the multiva-
riate Cox regression analysis were P ⬍ 0.1, or RR ⬍0.5 or ⬎2, in the
univariate analysis. A backward elimination procedure was then performed to
eliminate nonsignificant variables (P ⱖ 0.1).
Because IMD has been shown for the same patient cohort to be closely
correlated with the therapeutic outcome (21), this parameter was included in all
multivariate analyses. To obtain ORs and RRs comparable with the parameters
reported in the present study, data pertaining to this variable were grouped into
four categories, according to the number of microvessels/high power field: I,
0 – 80; II, 81–110; III, 111–130; and IV, ⬎130.
Statistical analyses were performed using the SPSS package (version 9.0.0;
SPSS, Inc., Chicago, IL).
RESULTS
Descriptive Statistics. A total of 98 patients were eligible for this
study. Patients and disease characteristics are presented in Table 1.
Most of the individuals had advanced T stage (88% T3– 4) or positive
N stage (66% N1–3); tumors of the tonsillar fossa (39%) and base of
tongue (30%) predominated. In 79 cases (81%), CR of the primary
tumor was achieved, and in 33 of the 65 patients (51%) with nodal
spread, CR of lymph node metastases was accomplished. In 42
Fig. 1. Immunohistochemical staining for HIF-1␣ in squamous cell cancers of the oropharynx. Expression at some distance from blood vessels (A, detail in B) or diffusely throughout
the entire tumor tissue (C, detail in D). A and C, ⫻64; B and D, ⫻170. Arrowheads point to blood vessels; ⴱ, necrosis.
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patients (43%), local failure occurred; this was because of either
relapse at the same site after CR (23 patients) or progression after
incomplete remission (19 patients). Fourteen patients developed dis-
tant metastasis.
In all HIF-1␣-positive tumor samples, immunostaining was nu-
clear; in rare instances, weak cytoplasmic reactivity was also ob-
served. Two predominant patterns of nuclear staining were encoun-
tered: focal expression, with the most intense reaction occurring distal
to the closest blood vessel and surrounding necrotic areas (Fig. 1, A
and B); and diffuse expression, independent of vessel proximity (Fig.
1, C and D). Predominant focal expression was found in 60 of 92
HIF-1␣-positive tumors (65%), whereas diffuse staining was encoun-
tered in 32 tumors (35%). Negative controls manifested no immuno-
reactivity. Six patients registered negative for HIF-1␣, 56 (57%)
exhibited weak staining (category I), and 36 (37%) manifested strong
immunoreactivity [category II (Table 1)]. In 17 of the 20 patients
(85%) with more than one biopsy/tumor, the immunoreactivity scores
of the biopsies were equal.
The only significant association between HIF-1␣ and patient or
disease characteristics was a reverse correlation between the immu-
noreactivity and histological grade (r ⫽ ⫺0.30; P ⫽ 0.01). The
distribution of HIF-1␣ staining between smokers and nonsmokers and
between patients receiving or not receiving chemotherapy was equally
balanced. No significant correlation existed between immunostaining
for HIF-1␣ and IMD (r ⫽ 0.07; P ⫽ 0.48).
Time-independent Outcome Analysis. In the univariate analysis,
HIF-1␣ was inversely correlated with both CR of the primary tumor
(OR⫽0.33; P ⫽ 0.03) and of lymph node metastases [OR, 0.34;
P ⫽ 0.02 (Table 2)]. IMD was predictive, as reported previously (21).
Of the four variables (age, T stage, HIF-1␣, and IMD) qualifying for
inclusion in the multivariate analysis of CR of the primary tumor,
HIF-1␣ (OR, 0.30; P ⫽ 0.01) and IMD (OR, 0.44; P ⫽ 0.03) retained
 HYPOXIA-INDUCIBLE FACTOR-1␣ IN RADIOTHERAPY
Fig. 2. Kaplan-Meier survival estimates for local failure-free survival, disease-free
survival, and overall survival, as pertaining to HIF-1␣ immunoreactivity.
their significance. Age and smoking habits were eliminated from the
model, with P ⱖ 0.1. Of the four variables (T stage, N stage, HIF-1␣,
and IMD) qualifying for inclusion in the multivariate analysis of CR
of lymph node metastasis, N stage (OR, 0.14; P ⫽ 0.002) and HIF-1␣
(OR, 0.30; P ⫽ 0.03) retained their significance. T stage and IMD
were removed from the model, with P ⱖ 0.1. In the univariate analysis
of distant metastasis, no variable attained statistical significance; but
T stage and HIF-1␣, having OR values of 2.21 and 2.71, respectively,
qualified for inclusion in the multivariate analysis. T stage was then
eliminated (P ⱖ 0.1), leaving HIF-1␣ with the same OR and border-
line significance (P ⫽ 0.07) as in the univariate analysis.
Time-dependent Survival Analysis. The univariate survival anal-
ysis revealed HIF-1␣ to be inversely correlated with local failure-free
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survival (P ⫽ 0.006, log rank), disease-free survival (P ⫽ 0.008, log
rank), and overall survival [P ⫽ 0.001, log rank (Fig. 2)]; the RRs
were 2.15, 2.01, and 2.17, respectively, per increase of one staining
grade (Table 3). The staining pattern (focal versus diffuse) had no
significant influence on local tumor control or survival (Tables 2 and
3). The correlation of local tumor control with IMD has been reported
previously (21). According to the inclusion criteria (P ⬍ 0.1 or
0.5 ⬎ RR ⬎ 2.0), T stage, smoking habits, HIF-1␣, and IMD qualified
for entry into the multivariate Cox regression model for all three
survival analyses. T stage, HIF-1␣, and IMD remained in the models
for local failure-free survival and disease-free survival, but smoking
habits were eliminated (P ⱖ 0.1). In the multivariate models, the RR
of HIF-1␣ increased to 2.43 for local failure-free survival
(P ⫽ 0.002), to 2.20 for disease-free survival (P ⫽ 0.004), and to 2.21
for overall survival (P ⫽ 0.0009), thereby establishing the independ-
ence of HIF-1␣ from T stage and IMD.
DISCUSSION
Prediction of the probability of successful treatment is of para-
mount importance for the individualization of therapy and the avoid-
ance of either unnecessary toxicity or treatment failure. Because
tumor hypoxia has been shown repeatedly to thwart the therapeutic
effects of ionizing radiation, considerable efforts have been invested
in the development of an easily applied means of measuring prethera-
peutic tumor oxygenation levels (5). The most promising results have
been achieved using polarographic oxygen electrodes. Readings thus
obtained confirmed the close relationship between tumor oxygenation
and radiation response in cancer of the uterine cervix (6, 7) and that
of the head and neck (8, 9, 22). Studies have focused on these two
tumor entities because the method is invasive. The need for specific
hypoxia markers that would be quantifiable either by noninvasive
imaging (positron emission tomography and single photon emission
computed tomography) or on routine tumor biopsies is therefore
obvious. Several immunologically detectable 2-nitroimidazoles,
which are metabolized predominantly in hypoxic areas, have been
proposed as potential candidates and tested in various animal and
human tumors (23), including squamous cell cancer of the head and
neck (24). However, the use of such agents involves their i.v. injection
prior to the removal of the biopsy, thus rendering necessary a second
intervention after the primary diagnosis.
In the present study, we investigated the predictive and prognostic
potential of HIF-1␣, which is a subunit of the HIF-1 heterodimer, a
protein that plays an essential role in oxygen homeostasis (13). Our
data indicate that HIF-1␣ is overexpressed in the neoplastic tissue of
the vast majority of squamous cell cancers of the oropharynx, a tumor
that is often treated by radical irradiation. Hypoxia is a principal
determinant of HIF-1␣ accumulation, and immunostaining for this
protein was encountered at some distance from tumor vessels (Fig. 1,
A and B), reflecting the decrease in oxygen concentration with in-
creasing distance from the capillaries. In other cases, however, im-
munoreactivity was observed diffusely throughout the entire tumor
tissue (Fig. 1, C and D). These latter findings may reflect the existence
of alternative regulatory modes of HIF-1␣ expression. The expression
of HIF-1␣ has been shown to be enhanced by v-src (25) and in
response to several growth factors, including insulin-like growth
factors 1 and 2, basic fibroblast growth factor, and epidermal growth
factor (26). Activation of the phosphatidylinositol 3-kinase/AKT/
FRAP pathway, which mediates signals from a broad range of growth
factors, has likewise been demonstrated to increase HIF-1␣ expres-
sion (27). In addition, p53 and the von Hippel-Lindau tumor suppres-
sor protein have been implicated in the degradation of HIF-1␣; the
loss of their function results in augmented HIF-1␣ levels (28, 29).
 HYPOXIA-INDUCIBLE FACTOR-1␣ IN RADIOTHERAPY

Table 2 Time-independent outcome analysis

CR of primary tumor CR of lymph node metastases Distant metastasis

OR CIa (95%) P OR CIa (95%) P OR CIa (95%) P

Univariate analysis
Age (yr), ⬍57/ⱖ57 0.41 0.14–1.18 0.10 0.88 0.33–2.31 0.79 0.95 0.31–2.95 0.94
T stage, T1–4 0.40 0.16–1.01 0.05 0.45 0.18–1.10 0.08 2.21 0.80–6.13 0.13
N stage, N0–3 0.80 0.49–1.30 0.37 0.14 0.04–0.48 0.002 1.27 0.73–2.21 0.40
Grade, G1–3 0.97 0.42–2.27 0.95 0.82 0.37–1.81 0.63 1.24 0.47–3.25 0.66
Smoking, no vs. yes 0.32 0.04–2.62 0.29 0.92 0.42–2.01 0.78 0.88 0.17–4.47 0.88
Chemotherapy, no vs. yes 1.49 0.45–5.00 0.52 1.36 0.49–3.77 0.55 1.10 0.31–3.84 0.89
HIF-1␣, 0–II 0.33 0.13–0.87 0.03 0.34 0.14–0.86 0.02 2.70 0.92–7.92 0.07
HIF-1␣ I, focal vs. diffuse 0.38 0.06–2.44 0.31 1.27 0.26–6.33 0.77 2.63 0.41–16.83 0.31
HIF-1␣ II, focal vs. diffuse 0.59 0.14–2.50 0.48 1.17 0.19–7.12 0.87 0.65 0.13–3.14 0.59
IMD, I–IVb 0.47 0.27–0.81 0.007 0.59 0.34–1.02 0.06 0.73 0.39–1.36 0.32
Multivariate analysis
Age (yr), ⬍57/ⱖ57 ⱖ0.1
T stage, T1–4 0.41 0.16–1.08 0.07 ⱖ0.1 ⱖ0.1
N stage, N0–3 0.14 0.04–0.49 0.002
Smoking, no vs. yes ⱖ0.1
HIF-1␣, 0–II 0.30 0.10–0.87 0.03 0.30 0.10–0.86 0.03 2.70 0.92–7.92 0.07
IMD, I–IV 0.44 0.24–0.82 0.01 ⱖ0.1
a
CI, confidence interval.
b
Data pertaining to IMD are adopted from Aebersold et al. (21).

We have demonstrated immunostaining for HIF-1␣ to be a power-
ful tool in predicting the success probability of radiotherapy in pa-
tients with squamous cell cancer of the oropharynx. To distinguish
between its predictive value in short- and long-term disease control,
CR of the primary disease and survival were analyzed separately.
Both in the univariate and multivariate analysis, an inverse correlation
was found to exist between HIF-1␣ and CR of the primary tumor
(Table 2). This association between HIF-1␣ immunoreactivity and
locoregional tumor control by radiotherapy was even more pro-
nounced when local failure-free survival was considered, with
P ⫽ 0.006 (Table 3). Again, the multivariate analysis confirmed
HIF-1␣ expression to be predictive, irrespective of the T stage or
IMD. In concert with this, HIF-1␣ and IMD were not correlated with
one another. The simultaneous application of hypoxic cell-specific
compounds, such as tirapazamine, represents a promising approach to
overcome hypoxia-induced radiation resistance (reviewed in Ref. 30).
Whether the assessment of HIF-1␣ expression can identify those
patients who would benefit most from such drugs remains to be
determined.
In addition to the association with a decreased local tumor control
rate, HIF-1␣ expression was significantly correlated with impaired
disease-free and overall survival (Fig. 2 and Table 3). Because lo-
coregional control is crucial for the overall survival of head and neck
cancer patients (31, 32), the prognostic power of HIF-1␣ staining lies
mainly in its association with local tumor control. However, the OR
for predicting distant metastasis according to HIF-1␣ expression was
2.71, with a borderline significance of P ⫽ 0.07 (Table 2). That P
failed to fall below 0.05 may reflect the weak statistical power of the
small patient group with distant metastasis (n ⫽ 14). Remarkably, all
patients with systemic disease manifested HIF-1␣ immunoreactivity
(Table 1), which supports the existence of a relevant association
between the expression of this factor and distant metastasis. Indeed, a
number of clinical studies have shown that low oxygen levels in
tumors are associated with increased metastasis (33, 34). Experimen-
tal studies suggest at least three possible explanations for these find-
ings:
(a) Hypoxia-induced cell death depends upon the presence of
wild-type p53 (35). Because cells with mutated p53 are far less
susceptible to hypoxia-induced apoptosis than those with the wild-
type form, intratumoral hypoxia will exert a strong selection pressure
for p53-mutated cells, thereby increasing the likelihood of tumor
progression.
(b) Hypoxia increases the mutation frequency (36).
(c) Hypoxia is a major stimulus for angiogenesis, which also
promotes distant metastasis (37).
The only significant correlation that existed between HIF-1␣ ex-
pression and patient or disease characteristics was a reverse associa-
tion with histological grade. Clinical studies using polarographic

Table 3 Survival analysis

Local failure-free survival Disease-free survival Overall survival
a a
RR CI (95%) P RR CI (95%) P RR CIa (95%) P
Univariate analysis
Age (yr), ⬍57/ⱖ57 1.13 0.62–2.08 0.69 1.06 0.60–1.88 0.84 0.89 0.52–1.50 0.65
T stage, T1–4 1.78 1.10–2.88 0.02 1.90 1.19–3.02 0.007 1.79 1.22–2.63 0.003
N stage, N0–3 0.99 0.74–1.33 0.96 1.00 0.76–1.31 0.98 1.23 0.96–1.58 0.11
Grade, G1–3 1.31 0.79–2.16 0.30 1.43 0.90–2.34 0.12 1.07 0.69–1.66 0.76
Smoking, no vs. yes 2.83 0.87–9.18 0.08 2.42 0.87–6.76 0.09 3.20 1.15–8.92 0.03
Chemotherapy, no vs. yes 0.89 0.45–1.77 0.74 0.77 0.39–1.51 0.44 1.14 0.63–2.05 0.66
HIF-1␣, 0–II 2.15 1.24–3.74 0.006 2.01 1.20–3.37 0.008 2.17 1.34–3.51 0.002
HIF-1␣ I, focal vs. diffuse 1.12 0.37–3.36 0.84 1.17 0.44–3.15 0.75 1.34 0.53–3.40 0.54
HIF-1␣ II, focal vs. diffuse 1.56 0.64–3.80 0.32 1.37 0.58–3.21 0.47 1.12 0.53–2.36 0.78
IMD, I–IVb 1.90 1.34–2.58 0.0002 1.71 1.25–2.32 0.0007 1.54 1.15–2.05 0.003
Multivariate analysis
T stage, T1–4 1.72 1.10–2.78 0.03 1.83 1.15–2.90 0.01 1.69 1.13–2.53 0.01
Smoking, no vs. yes ⱖ0.1 ⱖ0.1 3.56 1.08–11.74 0.04
HIF-1␣, 0–II 2.43 1.37–4.30 0.002 2.20 1.29–3.74 0.004 2.35 1.42–3.90 0.0009
IMD, I–IV 2.00 1.40–2.85 0.0001 1.79 1.28–2.50 0.0006 1.65 1.19–2.29 0.003
a
CI, confidence interval.
b
Data pertaining to IMD are adopted from Aebersold et al. (21).
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 HYPOXIA-INDUCIBLE FACTOR-1␣ IN RADIOTHERAPY
oxygen electrodes have failed to demonstrate the existence of a
relationship between hypoxia and histological grade, either in cancers
of the uterine cervix (7) or in those of the head and neck (38). The
significance of the association between HIF-1␣ and histological dif-
ferentiation thus remains to be clarified.
In the present study, we have shown that HIF-1␣ is overexpressed
in the vast majority of patients with squamous cell cancer of the
oropharynx and, even more important, that the degree of HIF-1␣
immunoreactivity has strong predictive and prognostic significance in
individuals treated by curative radiation therapy. The extent to which
hypoxia accounts for this association with treatment failure remains to
be clarified by simultaneous assessment of HIF-1␣ expression and
tumor oxygenation. Because HIF-1␣ expression is also induced by
tumor-specific genetic alterations (25–29), it may provide greater
prognostic information than measurement of intratumoral hypoxia.
ACKNOWLEDGMENTS
We thank Dr. P. H. Burri, Chairman of the Institute of Anatomy, University
of Berne, for his generous support, and B. de Breuyn, as well as B. Krieger for
technical assistance.
REFERENCES
1. Dachs, G. U., and Chaplin, D. J. Microenvironmental control of gene expression:
implications for tumor angiogenesis, progression, and metastasis. Semin. Radiat.
Oncol., 8: 208 –216, 1998.
2. Teicher, B. A. Hypoxia and drug resistance. Cancer Metastasis Rev., 13: 139 –168,
1994.
3. Mottram, J. C. A factor of importance in the radio-sensitivity of tumors. Br. J. Radiol.,
9: 606 – 614, 1936.
4. Moulder, J. E., and Rockwell, S. Tumor hypoxia: its impact on cancer therapy. Cancer
Metastasis Rev., 5: 313–341, 1987.
5. Raleigh, J. A., Dewhirst, M. W., and Thrall, D. E. Measuring tumor hypoxia. Semin.
Radiat. Oncol., 6: 37– 45, 1996.
6. Hockel, M., Knoop, C., Schlenger, K., Vorndran, B., Baussmann, E., Mitze, M.,
Knapstein, P. G., and Vaupel, P. Intratumoral pO2 predicts survival in advanced
cancer of the uterine cervix. Radiother. Oncol., 26: 45–50, 1993.
7. Fyles, A. W., Milosevic, M., Wong, R., Kavanagh, M. C., Pintilie, M., Sun, A.,
Chapman, W., Levin, W., Manchul, L., Keane, T. J., and Hill, R. P. Oxygenation
predicts radiation response and survival in patients with cervix cancer. Radiother.
Oncol., 48: 149 –156, 1998.
8. Gatenby, R. A., Kessler, H. B., Rosenblum, J. S., Coia, L. R., Moldofsky, P. J., Hartz,
W. H., and Broder, G. J. Oxygen distribution in squamous cell carcinoma metastases
and its relationship to outcome of radiation therapy. Int. J. Radiat. Oncol. Biol. Phys.,
14: 831– 838, 1988.
9. Nordsmark, M., Overgaard, M., and Overgaard, J. Pretreatment oxygenation predicts
radiation response in advanced squamous cell carcinoma of the head and neck.
Radiother. Oncol., 41: 31–39, 1996.
10. Wang, G. L., Jiang, B. H., Rue, E. A., and Semenza, G. L. Hypoxia-inducible factor
1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc.
Natl. Acad. Sci. USA, 92: 5510 –5514, 1995.
11. Jiang, B. H., Semenza, G. L., Bauer, C., and Marti, H. H. Hypoxia-inducible factor
1 levels vary exponentially over a physiologically relevant range of O2 tension. Am. J.
Physiol, 271: C1172–C1180, 1996.
12. Yu, A. Y., Frid, M. G., Shimoda, L. A., Wiener, C. M., Stenmark, K., and Semenza,
G. L. Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible fac-
tor-1 in the lung. Am. J. Physiol., 275: L818 –L826, 1998.
13. Semenza, G. L. Regulation of mammalian O2 homeostasis by hypoxia-inducible
factor 1. Annu. Rev. Cell Dev. Biol., 15: 551–578, 1999.
14. Wang, G. L., and Semenza, G. L. Purification and characterization of hypoxia-
inducible factor 1. J. Biol. Chem., 270: 1230 –1237, 1995.
15. Huang, L. E., Arany, Z., Livingston, D. M., and Bunn, H. F. Activation of hypoxia-
inducible transcription factor depends primarily upon redox-sensitive stabilization of
its ␣ subunit. J. Biol. Chem., 271: 32253–32259, 1996.
2916
Downloaded from cancerres.aacrjournals.org on August 23, 2018. © 2001 American Association for Cancer
Research.
16. Talks, K. L., Turley, H., Gatter, K. C., Maxwell, P. H., Pugh, C. W., Ratcliffe, P. J.,
and Harris, A. L. The expression and distribution of the hypoxia-inducible factors
HIF-1␣ and HIF-2␣ in normal human tissues, cancers, and tumor-associated macro-
phages. Am. J. Pathol., 157: 411– 421, 2000.
17. Zhong, H., De Marzo, A. M., Laughner, E., Lim, M., Hilton, D. A., Zagzag, D.,
Buechler, P., Isaacs, W. B., Semenza, G. L., and Simons, J. W. Overexpression of
hypoxia-inducible factor 1␣ in common human cancers and their metastases. Cancer
Res., 59: 5830 –5835, 1999.
18. Birner, P., Schindl, M., Obermair, A., Plank, C., Breitenecker, G., and Oberhuber, G.
Overexpression of hypoxia-inducible factor 1␣ is a marker for an unfavorable
prognosis in early-stage invasive cervical cancer. Cancer Res., 60: 4693– 4696, 2000.
19. Zagzag, D., Zhong, H., Scalzitti, J. M., Laughner, E., Simons, J. W., and Semenza,
G. L. Expression of hypoxia-inducible factor 1␣ in brain tumors: association with
angiogenesis, invasion, and progression. Cancer (Phila.), 88: 2606 –2618, 2000.
20. Lee, S. H., Wolf, P. L., Escudero, R., Deutsch, R., Jamieson, S. W., and
Thistlethwaite, P. A. Early expression of angiogenesis factors in acute myocardial
ischemia and infarction. N. Engl. J. Med., 342: 626 – 633, 2000.
21. Aebersold, D. M., Beer, K. T., Laissue, J., Hug, S., Kollar, A., Greiner, R. H., and
Djonov, V. Intratumoral microvessel density predicts local treatment failure of
radically irradiated squamous cell cancer of the oropharynx. Int. J. Radiat. Oncol.
Biol. Phys., 48: 17–25, 2000.
22. Brizel, D. M., Sibley, G. S., Prosnitz, L. R., Scher, R. L., and Dewhirst, M. W. Tumor
hypoxia adversely affects the prognosis of carcinoma of the head and neck. Int. J.
Radiat. Oncol. Biol. Phys., 38: 285–289, 1997.
23. Hodgkiss, R. J. Use of 2-nitroimidazoles as bioreductive markers for tumour hypoxia.
Anticancer Drug Des., 13: 687–702, 1998.
24. Evans, S. M., Hahn, S., Pook, D. R., Jenkins, W. T., Chalian, A. A., Zhang, P.,
Stevens, C., Weber, R., Weinstein, G., Benjamin, I., Mirza, N., Morgan, M., Rubin,
S., McKenna, W. G., Lord, E. M., and Koch, C. J. Detection of hypoxia in human
squamous cell carcinoma by EF5 binding. Cancer Res., 60: 2018 –2024, 2000.
25. Jiang, B. H., Agani, F., Passaniti, A., and Semenza, G. L. V-SRC induces expression
of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular
endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression.
Cancer Res., 57: 5328 –5335, 1997.
26. Feldser, D., Agani, F., Iyer, N. V., Pak, B., Ferreira, G., and Semenza, G. L.
Reciprocal positive regulation of hypoxia-inducible factor 1␣ and insulin-like growth
factor 2. Cancer Res., 59: 3915–3918, 1999.
27. Zhong, H., Chiles, K., Feldser, D., Laughner, E., Hanrahan, C., Georgescu, M. M.,
Simons, J. W., and Semenza, G. L. Modulation of hypoxia-inducible factor 1␣
expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/
FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis
and therapeutics. Cancer Res., 60: 1541–1545, 2000.
28. Ravi, R., Mookerjee, B., Bhujwalla, Z. M., Sutter, C. H., Artemov, D., Zeng, Q.,
Dillehay, L. E., Madan, A., Semenza, G. L., and Bedi, A. Regulation of tumor
angiogenesis by p53-induced degradation of hypoxia-inducible factor 1␣. Genes
Dev., 14: 34 – 44, 2000.
29. Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C.,
Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R., and Ratcliffe, P. J. The
tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-depen-
dent proteolysis. Nature (Lond.), 399: 271–275, 1999.
30. Rauth, A. M., Melo, T., and Misra, V. Bioreductive therapies: an overview of drugs
and their mechanisms of action. Int. J. Radiat. Oncol. Biol. Phys., 42: 755–762, 1998.
31. Vokes, E. E., Weichselbaum, R. R., Lippman, S. M., and Hong, W. K. Head and neck
cancer. N. Engl. J. Med., 328: 184 –194, 1993.
32. Beer, K. T., Greiner, R. H., Aebersold, D. M., and Zbaren, P. Carcinoma of the
oropharynx: local failure as the decisive parameter for distant metastases and survival.
Strahlenther. Onkol., 176: 16 –21, 2000.
33. Brizel, D. M., Scully, S. P., Harrelson, J. M., Layfield, L. J., Bean, J. M., Prosnitz,
L. R., and Dewhirst, M. W. Tumor oxygenation predicts for the likelihood of distant
metastases in human soft tissue sarcoma. Cancer Res., 56: 941–943, 1996.
34. Hockel, M., Schlenger, K., Aral, B., Mitze, M., Schaffer, U., and Vaupel, P. Asso-
ciation between tumor hypoxia and malignant progression in advanced cancer of the
uterine cervix. Cancer Res., 56: 4509 – 4515, 1996.
35. Graeber, T. G., Osmanian, C., Jacks, T., Housman, D. E., Koch, C. J., Lowe, S. W.,
and Giaccia, A. J. Hypoxia-mediated selection of cells with diminished apoptotic
potential in solid tumours. Nature (Lond.), 379: 88 –91, 1996.
36. Reynolds, T. Y., Rockwell, S., and Glazer, P. M. Genetic instability induced by the
tumor microenvironment. Cancer Res., 56: 5754 –5757, 1996.
37. Ellis, L. M., and Fidler, I. J. Angiogenesis and metastasis. Eur. J. Cancer, 32A:
2451–2460, 1996.
38. Becker, A., Stadler, P., Lavey, R. S., Hansgen, G., Kuhnt, T., Lautenschlager, C.,
Feldmann, H. J., Molls, M., and Dunst, J. Severe anemia is associated with poor
tumor oxygenation in head and neck squamous cell carcinomas. Int. J. Radiat. Oncol.
Biol. Phys., 46: 459 – 466, 2000.
Expression of Hypoxia-inducible Factor-1α: A Novel Predictive
and Prognostic Parameter in the Radiotherapy of
Oropharyngeal Cancer
Daniel M. Aebersold, Philipp Burri, Karl T. Beer, et al.

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