Int. J. Cancer: 116, 734–739 (2005)
' 2005 Wiley-Liss, Inc.
Overexpression of hypoxia-inducible factor HIF-1a predicts early relapse
in breast cancer: Retrospective study in a series of 745 patients
Jean-Philippe Dales1*, Stéphane Garcia1, Séverine Meunier-Carpentier1, Lucile Andrac-Meyer1, Olivier Haddad2,
Marie-Noölle Lavaut1, Claude Allasia1, Pascal Bonnier2 and Colette Charpin1
1
Department of Pathology, Hôpital Nord, Marseilles, France
2
Department of Oncologic Gynaecology, Hôpital de la Conception, Universite´ de la Me´diterrane´e (Aix-Marseille II),
Faculte´ de Me´decine, Marseilles, France
Hypoxia-inducible factor-1a (HIF-1a) is a transcription factor that
is involved in tumour growth and metastasis by regulating genes
involved in response to hypoxia. HIF-1a protein overexpression
has been shown in a variety of human cancers, but only 2 studies
have documented the prognostic relevance of HIF-1a expression
in breast cancer. The aim of our study was to determine accurately the impact of HIF-1a expression on prognosis in a large series (n = 745) of unselected patients with invasive breast cancer in
terms of overall survival, local recurrence and distant metastasis
risk. HIF-1a expression was investigated using immunohistochemical assays on frozen sections, and correlated with patients’ outcome (median follow-up = 13.5 years). Univariate (Kaplan-Meier)
analysis showed that high levels of HIF-1a expression (cutoff =
10%) significantly correlated with poor overall survival ( p =
0.019). HIF-1a expression correlated with high metastasis risk
among the whole group of patients ( p = 0.008). Multivariate analysis (Cox model) showed that the HIF-1a predictive value was independent of other current prognostic indicators. Moreover among
node negative ones, HIF-1a expression was also significantly predictive of metastasis risk ( p = 0.03) and of relapse (p = 0.035). All
the data suggest that HIF-1a is associated with a worse prognosis
in patients with invasive breast carcinoma. Furthermore HIF-1a
immunodetection may be considered as a potential indicator for
selecting patients who could benefit from specific therapies interfering with HIF-1a pathway.
' 2005 Wiley-Liss, Inc.
Key words: hypoxia-inducible factor; HIF-1a; prognosis; breast
carcinoma; immunohistochemistry
Neoangiogenesis is essential for tumour growth and the development of metastases. Cancer cell proliferation may outpace the
rate of angiogenesis, resulting in tissue hypoxia1 and cellular
adaptation to hypoxia is a key step in tumour progression.2 This
adaptation is regulated mainly by the hypoxia-inducible factor-1
(HIF-1) that is known to play an essential role in oxygen homeostasis.3–7 HIF-1 is a ubiquitously expressed heterodimeric transcription factor comprising an a and a b subunit. Three isoforms
of the a subunit have been identified: HIF-1a, HIF-2a (also
referred to as EPAS-1, MOP2, HLF and HRF) and HIF-3a. HIF1a is the best characterized isoform that is regulated by oxygen
levels and forms a heterodimer with a constitutive HIF-1b subunit
that is identical to aryl hydrocarbon nuclear receptor translocator
(ARNT).8,9 The amino-terminal half of each subunit contains
basic helix-loop-helix (bHLH) and PAS (Per-ARNT-/AhR-Sim)
motifs that are required for heterodimerization and DNAbinding.5,10
HIF-1a is the sole oxygen-regulated subunit that determines
HIF-1 activity.4,11,12 Hypoxic conditions lead to HIF-1a protein
stabilization,3 and thus the protein’s intracellular levels increase.
Once stabilized, HIF-1a translocates to the nucleus guided by a
nuclear localization signal present in C-terminus.13 After translocation HIF-1a heterodimerizes with HIF-1b, and the resulting
HIF-1 complex binds to an enhancer element called the hypoxiaresponse element (HRE) in oxygen-regulated target genes.3 The
amount of HIF-1a protein in the nucleus determines the functional
activity of the HIF-1 complex. HIF-1a alters the transcription of a
spectrum of genes mainly involved in erythropoiesis, angiogenesis
and glucose metabolism including erythropoietin, transferrin,
Publication of the International Union Against Cancer
endothelin-1, inducible nitric oxide synthetase, heme oxygenase1, vascular endothelial growth factor (VEGF), vascular endothelial
growth factor receptor-1 (VEGF-R1), insulin-like growth factor-2,
insulin-like growth factor-binding proteins and different glucose
transporters and glycolytic enzymes.14–17 Protein products of these
downstream genes function to increase oxygen delivery or to activate alternate metabolic pathways that do not require oxygen.
Interestingly, most of these proteins are implicated in tumour
progression.18
There is increasing evidence that HIF-1 is one of the key
factors in the progression of human malignant disease.19–21
Several studies have showed that HIF-1a protein was overexpressed in a variety of human cancers22 including lung, prostate, breast, colon cancer and direct correlations between HIF1a and tumour angiogenesis have been demonstrated.23 Only
a few reports, however, have provided evidence concerning
the impact of HIF-1a expression on the prognosis of human
breast carcinoma. Increased levels of HIF-1a have been
reported during breast carcinogenesis, especially in poorly differentiated lesions,24 and one more recent study has shown
the influence of HIF-1a protein expression on the behaviour
of node positive invasive breast carcinoma.25 In contrast
another recent study has demonstrated that HIF-1a expression
correlated with a worse prognosis in node negative but not in
node positive patients.26 These conflicting and preliminary
data in human breast cancer may result from either too short
follow-up or too small series. The practical relevance of HIF1a as prognostic indicator and potential target for specific
therapies in node negative patients then seemed to deserve
further investigation.
The aim of our study was to accurately document the variations
of HIF-1a expression in a large series of breast carcinomas
(n ¼ 745), and to correlate the immunohistochemical expression
of this marker on frozen samples with patients’ outcome in terms
of overall survival and metastasis- and recurrence-free survival
(long-term follow-up, median 13.5 years).
Material and methods
Materials
Seven hundred and forty-five patients from 25–79 years of age
(mean ¼ 56.1 years, SD ¼ 13.3) with breast carcinoma underwent
surgery from 1986–95. They did not receive chemotherapy or hormone therapy before surgery. The patients underwent axillary
node excision combined with wide local excision with margins
Grant sponsor: GEFLUC Marseille-Provence; Grant sponsor: French
Pathology Society; Grant sponsor: Université de la Méditerranée (AixMarseille II); Grant sponsor: Ministére de la santé et de la recherche (Cancéropôle PACA).
*Correspondence to: Faculté de Médecine Secteur Nord, Service
d’Anatomie et de Cytologie Pathologiques, Bd Pierre Dramard, 13916
Marseille Cedex 20 France. Fax: þ00-33-4-91-69-89-53.
E-mail: Jdales@mail.ap-hm.fr
Received 16 April 2004; Accepted after revision 24 November 2004
DOI 10.1002/ijc.20984
Published online 22 April 2005 in Wiley InterScience (www.interscience.
wiley.com).
EXPRESSION OF HiF-1a IN BREAST CARCINOMA
735
FIGURE 1 – Immunoperoxidase staining of cryostat tissue sections using HIF-1a polyclonal antibody in invasive breast carcinomas. (a) Strong
expression of HIF-1a is observed in a vast majority of tumour cells and in the tumour/stromal margin. (b) HIF-1a is sometimes expressed in a
heterogeneous pattern in the cytoplasm of tumour cells. (c,d) HIF-1a protein expression may be observed in a subset of stromal cells.
clearance or mastectomy in the Department of Oncologic Gynaecology in Conception Hospital, Marseilles. All the specimens were
examined in the same Department of Pathology by experienced
pathologists.
The patients’ follow-up ranged from 8–17 years. The 2003
records showed that 277 (37.2%) patients relapsed, among whom
191 (25.6%) died and 468 (62.8%) were disease-free. Overall survival was calculated as the period from surgery until date of death.
Metastasis-free survival was calculated as the period from surgery
until date of metastasis.
Mean tumour size was 20.7 mm (SD ¼ 13.8) and 23% tumours
were 10 mm large, 40% were >10 mm and 20 mm large, 22%
were >20 mm and 30 mm large, and 15% larger than 30 mm.
Histological examination of surgical specimens was carried out on
paraffin embedded sections stained with hematoxylin, eosin and
saffronin.
Tumours corresponded to ductal carcinomas (n ¼ 507, 68%),
lobular carcinomas (n ¼ 134, 18%), and to other types including
mucinous, medullary, papillary, apocrine or mixed carcinomas (n ¼ 104, 14%). Tumours were Grade 1 in 24% of these
cases (n ¼ 179), Grade 2 in 51% (n ¼ 380) and Grade 3 in 25%
(n ¼ 186). Tumor grading, initially assessed by using the grading
methods of Scarff et al.,27 was re-evaluated according to Elston
and Ellis.28
A mean of 14.7 (SD 6 4.3) lymph node was found in
axillary node excision and 372 (49.9%) patients were node
negative.
Immunostaining procedure and quantification
of HIF-1a expression
Fresh tissue fragments were sampled by pathologists immediately after intraoperative diagnosis. The fragment size varied
according to the tumour size (average ¼ 5 mm long, 4 mm wide,
3 mm thick). Fragments were obtained from dense tumour areas
that lacked grossly visible adipose tissue. They were then dipped
promptly in liquid nitrogen and stored at 808C in the laboratory tumour library. Immunodetection was carried out on 5-mm
thick sections (cryostat Leica CM 3050, Rueil Malmaison,
France).
Immunoperoxidase procedure was realized using polyclonal
rabbit (1:400 dilution) antihuman HIF-1a (H-206) (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) and Ventana Gene II device
with Ventana kits. Immunoreactivity of HIF-1a in breast cancer
tissue was determined by assessing semiquantitatively the percentage of decorated tumour cells by experienced pathologists using a
Zeiss Axioplan microscope as reported previously29–31 on the
whole tissue section by examining all optical fields. Staining
intensity was not incorporated in the scoring method because it
was more or less constant.
Statistical analysis
The Kaplan-Meier method was used to analyze disease-free
and overall survival rates. The difference between curves was
evaluated with the Mantel Cox test (or log-rank test) for observations regarding censored survival or events. All computations
736
DALES ET AL.
FIGURE 4 – p-Values curve (log-rank test) showing optimal cutoff
points for HIF-1a immunostaining for overall survival and recurrence
in all patients according to Altman et al.32
FIGURE 2 – Distribution of HIF-1a expression evaluated (%) in frozen sections from 745 patients with breast carcinoma.
FIGURE 5 – p-Values curve (log-rank test) showing optimal cutoff
points for HIF-1a immunostaining for recurrence in node negative
patients according to Altman et al.32
FIGURE 3 – Survivorship plot (Kaplan-Meier log-rank test) showing
overall survival (all patients) of 745 patients with breast carcinoma
with a higher ( p ¼ 0.019) risk of death for tumours with high expression levels of HIF-1a (10%).
TABLE I – OVERALL SURVIVAL OF PATIENTS CORRELATES WITH HiF-1a
EXPRESSION ON FROZEN SECTIONS
All patients (n ¼ 745)
Deceased
Alive
HIF-1a expression
<10%
>10%
39
163
152
391
p-value
0.019
—
were done with NCSS 2000 statistical software (Kaysville,
UT). HIF-1a expression was stratified and correlated with
major events during the course of the disease (distant metastasis or local recurrence) and with the overall survival rate to
define immunohistochemical thresholds of prognostic significance. The optimal HIF-1a cutoff point of positive staining
endowed with prognostic significance was determined after statistical validation.32 The effect of multiple factors on survival
was tested with a Cox multivariate proportional hazards mode
(NCSS 2000). The assumptions of proportional hazards were
evaluated by examining data on the log cumulative hazard that
FIGURE 6 – Kaplan-Meier univariate analysis showing higher risk
of metastasis ( p ¼ 0.008) in patients (all patients) with tumours with
high expression levels of HIF-1a (10%).
were stratified by the histoprognostic factors used in the model
(tumour size, histologic grade, nodal status) and by examining
residual data vs. survival time. All p-values were based on 2sided testing.
EXPRESSION OF HiF-1a IN BREAST CARCINOMA
FIGURE 7 – Kaplan-Meier univariate analysis showing higher risk
of metastasis (p ¼ 0.03) in node negative (N) patients with tumours
with high expression levels of HIF-1a (10%).
737
FIGURE 8 – Kaplan-Meier univariate analysis showing higher risk
of metastasis or recurrence ( p ¼ 0.015) in patients (all patients) with
tumours with high expression levels of HIF-1a (10%).
TABLE II – HiF-1a EXPRESSION CORRELATES WITH METASTASIS EVENT
IN ALL PATIENTS AND IN NODE NEGATIVE ONES
HIF-1a expression
All patients (n ¼ 745)
Metastasis
No metastasis
Node negative patients (n ¼ 372)
Metastasis
No metastasis
p-value
<10%
>10%
44
158
182
361
0.008
—
21
83
83
185
0.03
—
Results
HIF-1a distribution in tissue sections
The staining pattern exhibited a clear-cut delineation, with discriminative cell labeling and no background reactivity. HIF-1a
expression was observed in all samples although the proportion of
cells expressing HIF-1a varied considerably between tumours.
The spatial arrangement of HIF-1a expression indicated a heterogeneous distribution across the tumour area. HIF-1a expression
was essentially observed in the carcinomatous cells. HIF-1a
showed cytoplasmic reactivity with very weak nuclear reactivity
in some tumour cells (Fig. 1a,b). In some cases, HIF-1a immunostaining was located in stromal fibroblasts, endothelial cells and
macrophages (Fig. 1c,d). The staining intensity varied within a
given section and between sections. HIF-1a expression was semiquantitatively evaluated (percentage of decorated tumour cells).
The distribution of the HIF-1a levels is shown in Figure 2
(mean ¼ 16.32%, SD ¼ 7.98, median ¼ 14%).
Univariate (Kaplan-Meier/log-rank) analysis and HIF-1a
prognostic significance
HIF-1a levels (cutoff point ¼ 10%) correlated (p ¼ 0.019) with
overall survival (Fig. 3, Table I). Tumours with HIF-1a expression
levels higher than 10% were associated with a poorer survival as
compared to those that exhibited lower HIF-1a levels. The validation of the optimal cutoff point for HIF-1a levels is shown in Figures 4 and 5, from p-values curve.32 In node negative patients
subset, however, HIF-1a immuno expression did not retain a prognostic significance. Among the total group, HIF-1a levels >10%
correlated with early and widespread metastasis (p ¼ 0.008)
(Fig. 6). A similar correlation was also observed (p ¼ 0.03) in
node negative patients (Fig. 7, Table II). HIF-1a levels >10% were
correlated (p ¼ 0.015) with higher relapse risk (metastasis and
FIGURE 9 – Kaplan-Meier univariate analysis showing higher risk
of metastasis or recurrence ( p ¼ 0.035) in node negative (N)
patients with tumours with high expression levels of HIF-1a (10%).
local recurrence) (Fig. 8). A close correlation was also observed
(p ¼ 0.035) in node negative patients subset (Fig. 9, Table III).
Multivariate (Cox Model) analysis and HIF-1a prognostic significance
In multivariate analysis, HIF-1a expression proved to be a prognostic indicator exhibiting a predictive value independent of the
tumour size and tumour grade in terms of overall survival and
metastasis free survival in all patients. HIF-1a expression did not
remain a significant independent prognostic variable in node negative patients (Table IV).
Discussion
Tumour hypoxia is known to correlate with increased malignancy, potential for metastasis and poor patient prognosis in a
number of tumour types including breast cancer.33 The transcriptional complex hypoxia-inducible factor-1 (HIF-1) plays a crucial role in physiological adaptation to hypoxia and is frequently
activated in tumours. activation of HIF-1 is considered to support
738
DALES ET AL.
TABLE III – HiF-1a EXPRESSION CORRELATES WITH RELAPSE RISK
(METASTASIS AND LOCAL RECURRENCE) IN ALL PATIENTS
AND IN NODE NEGATIVE ONES
HIF-1a expression
All patients (n ¼ 745)
Relapse
No relapse
Node negative patients (n ¼ 372)
Relapse
No relapse
p-value
<10%
>10%
62
140
215
328
0.015
—
29
75
99
169
0.035
—
TABLE IV – PROPORTIONAL HAZARD REGRESSION, COX MODEL
Probability level
Overall survival (all patients)
HIF-1a
Histological grade
Tumor size
Metastasis free survival (all patients)
HIF-1a
Histological grade
Tumor size
Metastasis free survival (node negative patients)
HIF-1a
Histological grade
Tumor size
Disease free survival (all patients)
HIF-1a
Histological grade
Tumor size
Disease free survival (node negative patients)
HIF-1a
Histological grade
Tumor size
0.030
0.033
0.760
0.023
0.002
0.653
0.106
0.003
0.945
0.158
0.004
0.793
0.317
0.005
0.652
tumour growth through activation of anaerobic metabolism and
induction of angiogenesis that is due in part to increased VEGF
gene transcription.6,18,34 Immunostaining for the a subunit of HIF1 (HIF-1a) can be used to identify the extent of HIF-1 activation
in tumour tissues. A significant association between HIF-1a overexpression and patient mortality has been shown in tumours of the
brain,35 cervix,36 ovary,37 and in nonsmall cell lung,38 head and
neck,39 oropharynx,40 oesophageal41 and nasopharyngeal
carcinomas.42
Only 2 clinicopathological studies that focus particularly on the
prognostic relevance of HIF-1a expression in human breast carcinoma are available in the literature.25,26 One study found that
HIF-1a protein overexpression was associated with significantly
shorter overall survival in a series of 206 patients in advancedstage breast cancer only (5-year follow-up) evidenced by positive
lymph nodes.25 In contrast, the study published by Bos et al.26 in
a series of 150 patients showed that increased expression of HIF1a correlated with overall survival only in node negative tumours.
In this regard, these discrepant results deserve a deeper insight
into HIF-1a prognostic significance in human breast cancer that
would more accurately determine HIF-1a clinical relevance not
only in terms of prognosis but also for further development of specific antiangiogenic therapy targeting HIF1. The aim of our study
was to determine more accurately, in a series significantly larger
(n ¼ 745) than those reported previously, the impact of HIF-1a
protein expression on the prognosis of unselected patients with
invasive breast cancer with long term follow-up (median ¼ 13.5
years). We investigated HIF-1a expression on frozen sections
(Leica 3050) with automated immunodetection (Ventana Gene II),
which provides optimal conditions for antigen preservation and
for procedure standardization. Our results show that in univariate
analysis (Kaplan-Meier), greater immunocytochemical expression
of HIF-1a significantly correlated with a poor overall survival.
This result corroborates the study of Bos et al.26 and Schindl
et al.25 in which marked HIF-1a expression correlated with
overall survival of all patients. Our study failed, however, to
identify HIF-1a prognostic significance in terms of overall survival in a node-negative subset of patients of particular interest
for therapy monitoring. These results contrast with those published by Bos et al.26 These discrepant observations may be
explained in part by the different method of tissue preparation
(paraffin vs. frozen sections). Moreover, our results show that
HIF-1a overexpression correlated with early relapse (local
relapse and distant metastasis) in all patients but also in a
node-negative subset, data not previously reported suggesting
that HIF-1 pathway is also implicated in local tumour progression. Recent studies have related the expression of HIF-1a
with resistance to radiotherapy in carcinomas of oropharynx,40
oesophagus43 and the head and neck.44 From these observations
it can be hypothesized that HIF-1 activation induces angiogenic
activity that confers proliferation advantage in breast cancer
cells during postsurgical treatments such as radiotherapy. We
also observed that HIF-1a expression was predictive of metastasis risk in all patients and in the node negative subgroup.
This infers a sensitivity of HIF-1a expression to identify a subset of node negative patients who might benefit from more
aggressive postsurgical therapies.
Given the major role of HIF-1 activity in compensating for loss
of oxygen by increasing its availability or providing metabolic
adaptation of tumour cells to oxygen deprivation, the inhibition of
this particular activity might provide a basis for the development
of future therapeutic agents targeting HIF-1. The clinical relevance of targeting HIF-1 is suggested by mouse xenograft experiments in which the loss of HIF-1a activity through
pharmacological or gene-therapy means resulted in decreased
tumour growth and vascular density in tumours derived from
breast carcinoma cells.18,45–47 We have shown that a marked
immunohistochemical expression of HIF-1a on frozen sections
can predict prognosis, in terms of overall survival of breast cancer
patients. Our study also shows that HIF-1a expression has a weak
predictive significance in terms of metastatic risk and local recurrence in node-negative patients. Immunodetection of HIF-1a
might further serve as an indicator for future adjuvant therapies
specifically aiming at HIF-1 activation and downstream transcriptional targets.
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