Prognostic significance of Hypoxia-Inducible Factor 1 alpha(HIF-1 alpha) expression in serous ovarian cancer: an immunohistochemical study.

Abstract

The hypoxia-inducible factor (HIF) has emerged as an attractive target for cancer therapy. The few publications addressing the prognostic significance of Hypoxia-Inducible Factor 1 alpha (HIF-1 alpha) cellular expression in ovarian cancer produced contradictory findings which are not permissible to widely acceptable conclusions and clinical applications. Our study was designed to investigate this by including a comparatively large number of cases and by using a combination of antibodies to analyze immunohistochemically the expression of HIF-1 alpha.
One hundred (n = 100) neoplastic and 20 benign (controls) pathological samples from paraffin-embedded tissue were included. They were classified after surgery as stage I (n = 23) and stage III G3 (n = 55). Also 22 borderline serous adenocarcinoma patients and 20 benign controls were stained. The mean follow up was 3 years. Only patients with the diagnosis of serous carcinoma of stage III, G3 who received 6 cycles of postoperative TC (175-180 mg/m2 paclitaxel and carboplatin after calculating the area under the concentration curve) with complete medical records (n = 55) were selected for survival analysis. The survival analysis of the samples compared two groups after the patients were dichotomized by HIF-1 alpha final score to positive and negative.
The frequency of the nuclear expression of HIF-1 alpha in benign tumours was significantly lower (median: no expression) than in borderline and ovarian cancer tumours combined (p < 0.001). HIF-1 alpha expression in serous ovarian carcinoma was not stage dependent. The overall survival of patients with tumours that stained strongly for HIF-1 alpha was significantly shorter than that of patients with tumours that stained weakly or were negative for HIF-1 alpha (p = 0.01). Kaplan-Meier survival curves confirmed that HIF-1 alpha "positive" had decreased overall survival compared to HIF-1 alpha "negative" patients (p = 0.003) and this was an independent adverse prognostic factor (multivariable analysis p = 0.006). HIF-1 alpha "positive" patients displayed a shorter median progress free interval (PFI) (not statistically significant p > 0.05). Interestingly the overall PFI of the subgroup of patients that have undergone suboptimal cytoreduction at primary surgery (n = 21) with tumours that stained strongly for HIF-1 alpha was significantly worse than that of patients with tumours that stained weakly or were negative for HIF- 1 alpha (p = 0.03).
Our report confirms the prognostic value of HIF-1 alpha when restricted to poorly differentiated serous ovarian carcinoma. In addition it shows that this association is elusive, since it is not only methodology-related but it can be antibody-depended. There is adequate evidence to speculate that targeting HIF-1 alpha could improve the long-term prognosis of these patients In order to increase the overall sensitivity of the immunoassay, maintaining acceptable levels of specificity, a panel of antibodies should be used.

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BMC Cancer
Open Access
Research article
Prognostic significance of Hypoxia-Inducible Factor 1
alpha(HIF-1alpha) expression in serous ovarian cancer: an
immunohistochemical study
Alexandros Daponte*1, Maria Ioannou2, Ilias Mylonis3, George Simos3,
Marcos Minas4, Ioannis E Messinis1 and George Koukoulis2
Address: 1Department of Obstetrics & Gynaecology, University of Thessalia, Larissa, Greece, 2Department of Pathology, University of Thessalia,
Larissa, Greece, 3Laboratory of Biochemistry Department of Medicine and Institute of Biomedical Research and Technology (BIOMED), Larissa,
Greece and 4Department of Hygiene and Epidemiology, University of Thessalia, Larissa, Greece
Email: Alexandros Daponte* - dapontea@otenet.gr; Maria Ioannou - mioan@med.uth.gr; Ilias Mylonis - simos@med.uth.gr;
George Simos - simos@med.uth.gr; Marcos Minas - markosminas@hotmail.com; Ioannis E Messinis - messinis@med.uth.gr;
George Koukoulis - poroi@otenet.gr
* Corresponding author
Abstract
Background: The hypoxia-inducible factor (HIF) has emerged as an attractive target for cancer
therapy. The few publications addressing the prognostic significance of Hypoxia-Inducible Factor
1α (HIF-1α) cellular expression in ovarian cancer produced contradictory findings which are not
permissible to widely acceptable conclusions and clinical applications. Our study was designed to
investigate this by including a comparatively large number of cases and by using a combination of
antibodies to analyze immunohistochemically the expression of HIF-1α.
Methods: One hundred (n = 100) neoplastic and 20 benign (controls) pathological samples from
paraffin-embedded tissue were included. They were classified after surgery as stage I (n = 23) and
stage III G3 (n = 55). Also 22 borderline serous adenocarcinoma patients and 20 benign controls
were stained. The mean follow up was 3 years. Only patients with the diagnosis of serous
carcinoma of stage III, G3 who received 6 cycles of postoperative TC (175–180 mg/m2 paclitaxel
and carboplatin after calculating the area under the concentration curve) with complete medical
records (n = 55) were selected for survival analysis. The survival analysis of the samples compared
two groups after the patients were dichotomized by HIF-1α final score to positive and negative.
Results: The frequency of the nuclear expression of HIF-1α in benign tumours was significantly
lower (median: no expression) than in borderline and ovarian cancer tumours combined (p <
0.001). HIF-1α expression in serous ovarian carcinoma was not stage dependent. The overall
survival of patients with tumours that stained strongly for HIF-1α was significantly shorter than that
of patients with tumours that stained weakly or were negative for HIF-1α (p = 0.01). Kaplan-Meier
survival curves confirmed that HIF-1α "positive" had decreased overall survival compared to HIF-
1α "negative" patients (p = 0.003) and this was an independent adverse prognostic factor
(multivariable analysis p = 0.006). HIF-1α "positive" patients displayed a shorter median progress
free interval (PFI) (not statistically significant p > 0.05). Interestingly the overall PFI of the subgroup
of patients that have undergone suboptimal cytoreduction at primary surgery (n = 21) with
Published: 16 November 2008
BMC Cancer 2008, 8:335 doi:10.1186/1471-2407-8-335
Received: 30 December 2007
Accepted: 16 November 2008
This article is available from: http://www.biomedcentral.com/1471-2407/8/335
© 2008 Daponte et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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tumours that stained strongly for HIF-1α was significantly worse than that of patients with tumours
that stained weakly or were negative for HIF-1α (p = 0.03).
Conclusion: Our report confirms the prognostic value of HIF-1α when restricted to poorly
differentiated serous ovarian carcinoma. In addition it shows that this association is elusive, since it
is not only methodology-related but it can be antibody-depended. There is adequate evidence to
speculate that targeting HIF-1α could improve the long-term prognosis of these patients In order
to increase the overall sensitivity of the immunoassay, maintaining acceptable levels of specificity, a
panel of antibodies should be used.
Background
The hypoxia-inducible factor (HIF) is an alpha (α)/beta
(β) heterodimeric DNA binding complex and directs an
extensive transcriptional response involving the induction
of genes relevant to tumour progression, such as angio-
genesis, glucose/energy metabolism, cellular growth,
metastasis, and apoptosis [1,2]. HIF-1α has emerged as an
attractive target for cancer therapy [3,4].
HIF-1α protein expression in ovarian cancer was first
investigated using immunohistochemistry (IHC) by
Zhong [5]. Thereafter, HIF-1α protein overexpression was
shown in 54–69% of the cancerous specimens tested vs.
12.5–31.4% in non-cancerous ovarian tissues [6,7].
Birner investigated for the first time, by IHC, the relation-
ship of hypoxia-inducible factor 1α (HIF-1α) expression
with prognosis and on response to chemotherapy in epi-
thelial ovarian tumours. He concluded that HIF-1α pro-
tein overexpression alone has no impact on the prognosis
of ovarian cancer, whereas in a subgroup of patients with
concurrent overexpression of 1α and p53 protein, a signif-
icantly shorter overall survival was observed [6].
In a subsequent study, using RT-PCR, the expression level
of 1α had no relation with the survival of ovarian cancer
patients and it was also independent of age, clinical stage
and histological subtype [8].
However, two recent publications offered additional
information indicating a relationship between HIF-1α
expression and prognosis in ovarian carcinomas.
Nakai by using Western Blotting concluded that in 52
patients with sub-optimally resected stage III/IV tumours
and further treated with combination postoperative
chemotherapy Taxol, Carbo (TC), HIF-1α expression cor-
related with significantly better survival [9].
Osada in a recent IHC study (24 patients with ovarian car-
cinoma stage III/IV patients, 10 of them poorly differenti-
ated G3, followed by cisplatin-based chemotherapy)
showed that nuclear HIF1a immunoreactivity was an
independent marker of poor prognosis [10].
At this point, some of the findings appear to be contradic-
tory and not permissible to widely acceptable conclusions
and clinical applications. There may be several factors
responsible for the published data variability. Our study
was designed to address two of them by including a com-
paratively large number of cases and by using a combina-
tion of antibodies to analyze immunohistochemically the
expression of HIF-1α.
Methods
Patients and samples
One hundred (n = 100) neoplastic and 20 benign (controls)
pathological samples from paraffin-embedded tissue were
included. The explorative laparotomies, chemotherapies and
the treatment of patients included in this trial was carried out
in the University Hospital of Larissa-University of Thessalia
Greece. All the necessary informed consents as requested by
the Institutional Review Board (IRB) of the University Hos-
pital were given. They were classified after surgery as stage I
(n = 23) and stage III (n = 55), G3 and 22 borderline serous
adenocarcinoma patients and 20 benign controls. The mean
follow up was 3 years. At the time of the analysis, 13/55 were
deceased due to their disease. Due to the short follow up all
stage I and borderline patients were alive at the time of the
analysis. Fifty five stage III pathological samples from paraf-
fin-embedded tissue were included in the survival analysis.
Only patients with the diagnosis of serous carcinoma of stage
III, G3 who received 6 cycles of postoperative TC (175–180
mg/m2 paclitaxel and carboplatin after calculating the area
under the concentration curve (AUC) with complete medical
records were selected for survival analysis. All above-men-
tioned samples were obtained one per patient. Clinicopatho-
logic information was obtained from medical records.
Cancer patients were classified after a staging laparotomy
was performed (the most common initial surgical proce-
dure consisted of abdominal hysterectomy, bilateral salp-
ingo-oophorectomy, omentectomy, and lymph node
sampling). The surgery was classified as complete when <
1 cm of tumor was left behind.
All slides were reviewed by two pathologists (G.K, M.I).
The surgical procedures were carried out in the Depart-

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ment of Obstetrics and Gynaecology University of Thes-
salia.
In order for a sample to be given a "positive" final score
convincing and easily detectable nuclear staining should
be seen with at least 3 out of the 5 HIF-1α antibodies
tested. Otherwise it was classified as negative.
The stage, grading, histology, age, family history, Ca 125
and level of cytoreduction (complete versus incomplete)
were reported.
Different regimens were given as second- and third-line
therapy, and the different groups (after further classifying
according to second-line chemotherapy) had small num-
bers to be analyzed separately. Therefore, although both
the progress-free interval (PFI) and the overall survival
were calculated, we report PFI after first-line chemother-
apy (TC) as indicative of the response to the first-line
chemotherapy. The survival analysis of the samples com-
pared two groups after the patients were dichotomized by
HIF-1α final score to positive and negative.
Immunohistochemical staining
Immunostaining was performed with the antibodies
listed in Table 1. The tissue samples had all been fixed in
10% buffered formalin, processed and embedded in par-
affin routinely. Sections were cut at 3 μm using a Leica
TP1020 microtome and dried overnight at 60°C. After
deparaffinization in xylene, the sections were rehydrated
in decreasing ethanol solutions and incubated in 0.3%
hydrogen peroxide for 10 min, to block endogenous per-
oxidase.
Different methods of antigen retrieval were tested in pilot
experiments (data not shown). Under the conditions of
the study, optimal antigen retrieval was achieved by
microwaving tissue sections in 0.01 M citrate buffer solu-
tion (pH 6) for 20 min, (LG WAVEDOM, 850 Watt). This
antigen retrieval remained optimal irrespectively of the
primary antibody type applied. After the antigen retrieval,
the sections cooled and washed in phosphate-buffered
saline (PBS) for three times. Tissue sections were incu-
bated overnight at 4°C with each antibody. The optimal
dilutions were determined with pilot experiments (data
not shown) see Table 1. Then, the slides were washed in
PBS and Envision fluid (polymer-peroxidase method,
EnVision+/HRP, DAKO, Denmark) was added, followed
by incubation for 30 min. Bound antibodies were visual-
ized by using 0.05% 3,3'-diaminobenzidine solution
(DAB solution, DAKO). Finally, sections were counter-
stained with hematoxylin and mounted in Entellan
(Merck, Germany). A detailed description of the Univer-
sity of Thessaly antibody has been included in a recently
published HIF-1α separate study [11].
Statistical analysis
Group comparisons were based on Spearman's test for
nominal variables (age, Ca125) and on the linear x2-test
for ordinal variables (stage, grading, complete/incom-
plete cytoreduction). The HIF-1α scores of the different
groups of patients were compared using the non-paramet-
ric test for multiple comparison (Kruscal Wallis-Mann
Whitney) followed by Dunn's test, which generalizes the
Bonferroni adjustment.
The factors possibly influencing the PFI and survival (age,
Ca 125, stage, HIF-1α scores) were determined by binary
logistic regression analysis using forward likelihood ratio
method.
From 55 poorly differentiated serous ovarian carcinomas,
33 tumour samples were classified as HIF-1α protein
"positive" (60%%) and 22 as HIF-1α protein "negative"
as they did not stain at all (40%). After the patients were
dichotomized by HIF-1α expression final score as positive
or negative (used as gold standard) Cohen's kappa statis-
tics were used to evaluate the measure of agreement kappa
( value) for HIF-1α protein detection between the differ-
ent antibodies.
Table 2: Multivariable Analysis for Hif-1a expression for Survival,
PFI and in patients with incomplete surgery
CoefficientP
Multivariable Analysis for Survival
Age
HIF
Ca125
constant
Multivariable Analysis for PFI
Age
HIF
Ca125
constant
Multivariable Analysis for PFI (-) incomplete
Age
HIF
Ca125
constant
-0.57702
20.42544
0.00316
61.99826
> 0.05
< 0.01
> 0.01
-0.60288
14.14634
0.00606
49.06490
> 0.05
> 0.05
> 0.05
-0.07887
13.56907
0.00606
8.71984
> 0.05
0.05
< 0.05
Multivariable Analysis for Survival
Table 1: Antibodies to HIF-1α used in this study*
AntibodyOptimal dilution Source
H1a671:200
1:200
1:20
1:75
1:200
Abcam
Santa Cruz
BD Biosciences
Neomarkers
Univ. Thessaly
Rabbit Polyclonal
54/HIF-1α
H1a67
Rabbit polyclonal

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A – Serous carcinoma
Figure 1
A – Serous carcinoma. Immunostaining for HIF1α with polyclonal antibody made in the University of Thessaly. B – Serous
carcinoma. Immunostaining for HIF1α with rabbit polyclonal antibody (Santa Cruz).note variable cytoplasmic staining in addi-
tion to the variable nuclear staining. C – Serous carcinoma. Immunostaining for HIF1α with monoclonal antibody 54/HIF-1α
(BD Biosciences). Note focal nuclear staining and lack of cytoplasmic staining. D – Serous carcinoma. Immunostaining for
HIF1α with monoclonal antibody H!a67 (Neomarkers). Note focal nuclear staining and lack of cytoplasmic staining. E – Serous
carcinoma. Immunostaining for HIF1α with monoclonal antibody H1a67 (Abcam). Note focal and weak nuclear staining and
lack of cytoplasmic staining in this case. Cytoplasmic staining was seen in other cases. F – Serous tumor of low malignant
potential. Note weak staining in several nuclei and focal intense staining of the nuclei at the tip of the papilla (see arrow and
insert). Immunostaining with clone H1a67 (Abcam).
A
C
E
B
D
F

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Survival analysis HIF(+) vs HIF (-)
Figure 2
Survival analysis HIF(+) vs HIF (-).

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Uni-variable and multivariable analyses were performed.
The covariates age, Ca 125, HIF-1α scores were used for
the multivariable analysis (in the 55 stage III, G3, serous
adenocarcinoma patients). Progression-free survival and
overall survival were estimated by Kaplan and Meier's
method. The log-rank test was used to compare differ-
ences between survival curves. The Cox regression was
used to calculate hazard ratios [12].
These were materialized using GraphPad Prism version 5
and SPSS version 15 software. All P values calculated are
two sided. P < 0.05 was considered to be significant.
Results
HIF-1α protein expression, (nuclear unless otherwise
specified), was shown in 14% of the non-cancerous ovar-
ian samples, in 53% of the borderline and in 60% of the
cancerous samples tested. The frequency of the nuclear
expression of HIF-1α in benign tumours differed signifi-
cantly (median: no expression) than in borderline and
ovarian cancer tumours combined (p < 0.001). The fre-
quency of the nuclear expression of HIF-1α in carcinomas
was higher than that of borderline tumours, but this dif-
ference was not significant (p > 0.05).
In the subset of 55 poorly differentiated serous ovarian
carcinomas the average patients' age was 62.5 and there
was no statistical difference between HIF-1α positive and
negative patients in regard to age, Ca 125, and complete/
incomplete cytoreduction (p > 0.05). Thirty three (33)
tumour samples were classified as HIF-1α protein "posi-
tive" (60%%) and 22 as HIF-1α protein "negative" as they
did not stain at all (40%). Representative immunohisto-
chemical findings are shown and commended in Figure 1.
HIF-1α expression level was independent of clinical stage
(p > 0.05).
Relationship between HIF-1 protein expression and
survival in patients with serous stage III poorly
differentiated adenocarcinoma
In the 55 patients with stage III, G3 serous carcinomas, the
overall survival of patients with tumours that stained
strongly for HIF-1α differed significantly than that of
patients with tumours that stained weakly or were nega-
tive for HIF-1α (p < 0.05). HIF-1α positive patients dis-
played a median survival of 28 months (18–43 months)
versus 39 months (range 15–73 months) for HIF-1α neg-
ative patients (Figure 2). Additionally, Kaplan-Meier sur-
vival curves confirmed that HIF-1α positive stage III, G3
patients had decreased overall survival compared to HIF-
1α negative patients (p < 0.01). Cox regression analysis
demonstrated that HIF-1α protein had a hazard ratio
(HR) of 3.853 (1.544 to 9.614) (Figure 2). Additionally
increased HIF-1α protein expression was an independent
adverse prognostic factor for survival (see multivariable
analysis in Table 2, p < 0.01).
Relationship between HIF-1 protein expression and
progression-free interval (PFI) in serous stage III poorly
differentiated adenocarcinomas
HIF-1α positive patients displayed a median PFI of 19
months (range 6–67 months) versus 20 months (8–42
months) in HIF-1α negative patients (not statistically sig-
nificant, p > 0.05). Additionally, Kaplan-Meier survival
curves showed that HIF-1α protein positive stage III, G3
cases had a decreased overall PFI compared to HIF-1α
negative patients but this was not statistically significant
(p > 0.05). Cox regression analysis demonstrated that
HIF-1α had an HR of 1.185 (0.5617 to 2.499). Increased
HIF-1α protein expression was not an independent
adverse prognostic factor for PFI (see multivariable analy-
sis in Table 2, p > 0.05).
Relationship between HIF-1 protein expression and PFI in
serous stage III poorly differentiated adenocarcinoma
patients with suboptimal cytoreduction
We performed a separate analysis in patients (n = 21) that
have undergone suboptimal cytoreduction at primary sur-
gery. The overall PFI of this subgroup of patients with
tumours that stained strongly for HIF-1α differed signifi-
cantly than that of patients with tumours that stained
weakly or were negative for HIF-1α (p < 0.05). HIF-1α
positive patients displayed a median PFI of 12 months
(range 6–24) versus 19.5 months (12–42) in HIF-1α neg-
ative patients. Additionally, Kaplan-Meier survival curves
showed that HIF-1α positive stage III, G3 patients had a
decreased overall PFI compared to HIF-1α negative
patients, and this was statistically significant (p < 0.05)
(Figure 3). Cox regression analysis demonstrated an HR of
2.88 (1.007 to 8.37) for HIF-1α. Increased HIF-1α expres-
sion was an independent adverse prognostic factor for PFI
for these group of patients (see multivariable analysis in
Table 2, p = 0.05).
Table 3: Kappa values (κ value 95% confidence interval) of antibodies to HIF-1α used in this study*
Antibody Optimal dilution Source
κ value (95% confidence interval)
54/HIF-1α
Rabbit polyclonal
Rabbit Polyclonal
H1a67
H1a67
1:20
1:200
1:200
1:75
1:200
BD Biosciences
Univ. Thessaly
Santa Cruz
Neomarkers
Abcam
0.754 (0.435–1.073)
0.634 (0.368–0.901)
0.618 (0.282–0.955)
0.569 (0.287–0.851)
0.435 (0.126–0.744).

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Relationship between HIF-1 nuclear or cytoplasmic
expression and PFI and survival in serous stage III poorly
differentiated adenocarcinoma
The group of HIF-1α positive samples (n = 33) were fur-
ther subdivided according to the presence of nuclear or
cytoplasmic immunostaining. The patients with samples
showing combined nuclear and cytoplasmic HIF-1α stain-
ing (n = 14) displayed a median survival of 38 months
(24–73) and a median PFI of 18 months (6–28). The ones
without cytoplasmic staining and with nuclear only stain-
ing (n = 19) displayed a median survival of 39 months
(15–69) and a median PFI of 24.5 months (6–67). When
these groups were compared, they did not differ signifi-
cantly for PFI (p > 0.05) or survival (p > 0.05).
Relationship between HIF-1 nuclear staining with
different antibodies in serous stage III poorly differentiated
adenocarcinoma patients
We used Cohen's κ statistics to compare the value of each
of the antibodies as probes capable of categorizing each
case as expressing (i.e. positive) or not expressing (nega-
tive) nuclear HIF-1α. As reported in the Methods section,
a "positive" designation required easily-detectable immu-
noreactivity with three out of five antibodies. A sample
showing nuclear immunoreactivity using one or two anti-
bodies would be designated as "negative". A high κ value
compared to HIF-1α positive patients would indicate that
a given antibody performed better in detecting positive
cases when compared with lower κ value antibodies. The
κ values (95% confidence intervals) for the different anti-
bodies are presented in ranking order in Table 3. The anti-
body with the lowest kappa value was H1a67-Abcam.
Birner et al. used this antibody in his IHC study and
reported that HIF-1α overexpression had no impact on
the prognosis [6]. To verify this, we performed a separate
survival analysis using only the findings from this anti-
body (Figure 4) and the survival analysis was not signifi-
cant, as Birner et al. had reported [6].
Discussion
HIF-1α expression has been noted in many different
tumor types and has been variably associated with adverse
prognosis. In ovarian carcinomas, a role for HIF-1α as an
adverse prognostic factor has been suggested but there are
conflicting findings [10]. Data variation can be ascribed to
many factors, including methodological differences or
statistical discrepancies due to the small size of patient
study groups. We observed that the frequency of nuclear
expression of HIF-1α in benign tumours was lower than
in borderline and ovarian cancer tumours, which is in
agreement with previous findings [6,7,10] and supports
the assertion that HIF-1α can be considered a hallmark of
tumour progression in ovarian carcinomas.
The frequency of nuclear expression of HIF-1α in stage III
carcinomas was higher than in stage I tumours, but this
was not statistically significant. However two points must
be emphasised. First we included only poorly differenti-
ated serous carcinomas in the survival analysis of our
study and second we observed high HIF-1α expression in
almost 50% of the stage I cases (12/25). Nakayama et al.,
using RT-PCR, also reported that HIF-1α expression level
was independent of clinical stage [8]. However, Osada et
al. reported that HIF-1α nuclear expression was signifi-
cantly higher in stage III and IV tumours than in those of
stages I and II [10]. Different histological types of ovarian
carcinoma were used in his study, and HIF-1α immunos-
taining was observed in only 12 of the 48 cases of stages I
and II (25%). In agreement with our data, Osada et al.
also reported higher expression in serous neoplasms
reporting that the role of the HIF pathway in ovarian can-
cer might be different among tumour subtypes [10].
In the 55 patients with stage III, G3 serous carcinoma in
this study, the overall survival of patients with tumours
that stained strongly for HIF-1α was significantly shorter.
Progress free interval) PFI_survival analysis in HIF(+) vs HIF (-) patients with incomplete cytoreductive surgery
Figure 3
Progress free interval) PFI_survival analysis in HIF(+)
vs HIF (-) patients with incomplete cytoreductive
surgery.
HIF (-)
HIF (+)
0
10
20
30
40
50
P=0.0383
Months
0 1020 3040 50
0
50
100
HIF (-)
HIF (+)
PFI
Percent survival

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This is in accordance with the recent in vivo IHC study by
Osada et al. [10], but contradicts the previous studies by
Birner et al. [6] and Nakayama et al. [8]. A possible expla-
nation is the difference in adjuvant chemotherapy.
Since we noted that HIF-1α assessment can be antibody-
dependent, a prudent future approach would be to use a
panel of antibodies in order to increase the overall sensi-
tivity of the immunoassay, at the same time maintaining
acceptable levels of specificity as we did in our study. This
could be achieved with the use of an affinity-purified pol-
yclonal antibody and one or two well-characterised mon-
oclonal antibodies. In future studies, additional
validation of the immunoassay could be performed by
including downstream targets of HIF-1α as their upregula-
tion is due to HIF-1α pathway activation [4]. Alterna-
tively, if there is an established correlation between HIF-
1α expression and a clinical variable, the performance of
the antibody could be tested by its potential to reproduce
the aforementioned association.
Our results suggest that the difference in progress free
interval is closely related to the chemoresponse of postop-
erative chemotherapy. One interpretation is that high
HIF-1α-expressing suboptimally resected tumours are
chemoresistant. It has been previously reported that
expression of HIF-1α may be predictive of responsiveness
to adjuvant therapy and radiotherapy [13-15]. Previous
studies showed that tumour hypoxia could lead to chem-
oresistance directly, due to the lack of oxygen availability,
and indirectly due to the alteration in gene expression and
subsequent changes in angiogenesis and pH changes [16].
Since HIF-1α is the key molecule regulated by tumour
hypoxia, HIF-1α deregulation in tumour cells may confer
resistance in these cells [17,18]. However, it was recently
reported using Western Blot analysis that HIF-1α-express-
ing ovarian tumours had a significantly higher rate of
response to postoperative TC chemotherapy and exhib-
ited significantly better survival [9]. The suggested expla-
nation was that taxanes could be effective in down-
regulating HIF-1α protein via effects on the microtubule
cytoskeleton that are correlated with HIF-1α mRNA trans-
lation [19].
Cytoplasmic HIF-1α expression is more prominent in
serous and endometrioid carcinoma, however this stain-
ing pattern is unexpected, since HIF-1α should translocate
from the cytoplasm to the nucleus in order to activate its
target genes within a short period of time, and accumula-
tion of cytoplasmic HIF-1α should not be detected [20].
However, one study showed that there is a HIF-1α variant
that is stable even in normoxia and does not translocate to
the nucleus under hypoxic conditions [21]. This splice
variant corresponds to the N-terminal part (aa 1–516) of
the wild-type 826 aa long HIF-1a while most of the anti-
bodies used in this study were raised against epitopes in
the C-terminal part of HIF-1a. This plus the low relative
expression of the variant make it rather unlikely that the
observed cytoplasmic staining is due to the presence of
this particular variant. Studies in other organ cancers have
also reported that immunolocalization of HIF-1α is not
limited inside the nucleus [22-24]. Our results, obtained
by log-rank test, showed that the prognosis was poorer in
patients with nuclear HIF-1α immunostaining. However,
immunostaining for cytoplasmic HIF-1α was associated
with shorter PFI and similar survival, but this was not sta-
tistically significant, which agrees with a recent study by
Osada et al. [10].
In this respect it should be noted that as we reported pre-
viously nuclear localization of HIF-1α requires its modifi-
cation by p42/44 MAPK [25] Therefore, exclusive nuclear
Progress free interval and overall survival curves (p > 0.05) using only Abcams antibody to define HIF1a positive/negative
Figure 4
Progress free interval and overall survival curves (p >
0.05) using only Abcams antibody to define HIF1a
positive/negative.
0 2040
DFI
6080
0
50
100
150
HIF (+)
HIF (-)
Percent survival
Surv N Abcam:Survival proportions
0 20 4060 80
0
50
100
150
HIF (+)
HIF (-)
P=ns
Time
Percent survival

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BMC Cancer 2008, 8:335http://www.biomedcentral.com/1471-2407/8/335
Page 9 of 10
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immunostaining of HIF-1α could indicate an activated
MAPK pathway and, subsequently, increased cellular pro-
liferation.
We observed that the median PFI of HIF-1α positive
patients was shorter but not statistically significant. How-
ever, in the subgroup of patients with suboptimally
cytoreduced tumours, the PFI of patients with tumours
that stained strongly for HIF-1α was significantly worse
than that of patients with tumours that stained weakly or
were negative for HIF-1α, and in those patients it proved
to be an independent prognostic factor. This is in accord-
ance with previous studies which showed that tumour
hypoxia could lead to chemoresistance directly [16].
The HIF system arises as an important molecular target in
the treatment of ovarian carcinoma. Staining the tumour
tissue obtained from the primary laparotomy with HIF 1a
antibodies could result in grouping them in positive or
negative as in our study, HIF 1a positive patients could
enter clinical trials using a number of agents that inhibit
HIF-1α accumulation including topotecan [26], 2-meth-
oxyestradiol [27,28] and the Hsp90 inhibitors [29,30].
Furthermore using the experimental models to test the
efficacy of rapamycin in ovarian cancer treatment, a signif-
icant correlation between HIF-1α inhibition and VEGF
down-regulation or increase of apoptosis has been dem-
onstrated [31], and it was mentioned that rapamycin
delays the tumour onset and progression [32]. Additional
effects were found to be exerted when rapamycin is
administered in combination with paclitaxel [31] and
tamoxifen [33] and could also be investigated in clinical
trials.
Clearly, additional studies are needed, however, we
strongly support determination of HIF-1-α expression by
immunohistochemistry in serous ovarian cancer for devis-
ing subgroups for individualized treatment regimens.
Conclusion
HIF-1α expression has been noted in many tumours and
it has been variably associated with adverse prognosis.
Our report confirms the prognostic value of HIF-1α in
serous ovarian cancer in a specific, albeit large, subset of
patients. In addition, we show that this association is elu-
sive, since it is not only methodology-related but it can be
antibody-dependent. From our observations, nuclear HIF-
1α expression might represent an important biological
marker in the evaluation of the prognosis of patients with
poorly differentiated serous ovarian carcinoma.
In our study, HIF-1α was an independent prognostic fac-
tor of survival and could be used to assist in decisions for
adjuvant therapies.
Furthermore, in designing a HIF-1α-targeting clinical trial,
it would be important to optimize the assessment of HIF-
1α expression by using a panel of antibodies to accurately
identify subgroups of ovarian carcinoma patients who
could benefit from novel HIF-1α-inhibiting therapeutic
strategies.
Abbreviations
HIF: Hypoxia-inducible factor; HIF-1α: Hypoxia-Induci-
ble Factor 1α; IHC: Immunohistochemistry; PFI: Progress
free interval; TC: Taxol, Carbo; AUC: Area under the con-
centration curve
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AD designed the study and drafted the manuscript. He
was involved as primary surgeon or first assistant in all the
patients surgery, was responsible for patients manage-
ment during their stay in the hospital and participated in
the muldisciplinary meeting regarding adjuvant therapy
and was responsible for their clinical follow up through
the gynaecological oncology outpatients clinic. MI gave
the pathology report in many cases, reviewed all the
pathology reports for the purpose of the study and col-
lected tumor specimens, organized the stainings, scored
sections and contributed to the design of the study and
the drafting of the manuscript. IM gave his technical
assistance in the laboratory. GS organized the stainings,
contributed to the design of the study and the drafting of
the manuscript. IMM, conducted the statistical analyses,
prepared figures and tables and helped draft the manu-
script. IEM performed as primary surgeon patients surgery
and contributed to the drafting of the manuscript. GK
reviewed all the pathology reports for the purpose of the
study, contributed to tumor specimens selection and scor-
ing and organised the setup of the ovarian carcinoma
database. He contributed to the design of the study and
the drafting of the manuscript. All authors read and
approved the final manuscript.
Acknowledgements
We thank the Dept. of Anatomic Pathology (Head: Professor George Kou-
koulis) and Maria Netsika for carrying out the immunohistochemical stain-
ings.
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