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Original Articles
Medical Biophysics Department, British Columbia Cancer Agency Research Centre, Vancouver, British Columbia, Canada
2
Department of Radiation Oncology, University of North Carolina, Chapel Hill, North Carolina, USA
3
Department of Microbiology and Molecular Genetics, University of California, Irvine, California, USA
Background: Although tumor hypoxia has been associated with a more aggressive phenotype and lower
cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the
chemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF-1a and
CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced
carcinoma of the cervix.
Methods: Two biopsies were taken one day after administration of pimonidazole and were analyzed for
pimonidazole binding using ow cytometry or immunohistochemistry. CAIX and HIF-1a expression and
degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were
examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF1a expression over the course of treatment.
Results: Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1a, or CAIX. The
CAIX positive fraction correlated with the pimonidazole positive fraction (r = 0.60). Weaker but signicant
correlations were observed between pimonidazole and HIF-1a (r = 0.31) and CAIX and HIF-1a (r = 0.41).
Taking the extent of marker colocalization into consideration increased the condence that all markers
were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with
the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the
hypoxic fraction measured using the three hypoxia markers. HIF-1a levels tended to decrease with time after the start of therapy.
Conclusions: Endogenous hypoxia marker binding shows reasonable agreement, in extent and location,
with binding of pimonidazole. CAIX staining pattern is a better match to the pimonidazole staining pattern
than is HIF-1a, and high CAIX expression in the absence (or low levels) of HIF-1a may indicate a different
biology. q 2006 International Society for Analytical Cytology
Key terms: hypoxia markers; oxygen electrode; CAIX; HIF-1a; pimonidazole; cervical cancer
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JANKOVIC ET AL.
METHODS
Patient Selection and Treatment Protocol
Ethical approval for this study was granted by the British
Columbia Cancer Agency Ethics Board and the University
of British Columbia Ethics committee. Over the course of
the study, more than 100 patients received pimonidazole
as an intravenous infusion before the study was closed at
47
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JANKOVIC ET AL.
FIG. 1. Analysis of hypoxia marker binding in antibody-stained sections and single cells. Panel (a) show a representative tiled tumor section stained for CAIX. In panel (b), this is converted to a gray scale
image. Panel (c) shows the total area of the section and panel (d) shows
the marker positive regions. The ratio of the number of red pixels in (d)
divided by the number of red pixels in (c) gives the percentage of the tumor that is CAIX positive.
40.3 (0.089.0)
38.5 (2.087.0)
4.1 (0.630.4)
4.8 (0.418.0)
4.4 (0.022.6)
5.7 (0.223.0)
4.3 (0.627.8)
6.1 (0.420.0)
5.2 (0.328.1)
4.2 (0.227.8)
66.0 (0.0100.0)
54.5 (2.0100.0)
42.0 (3.089.0)
37.0 (0.087.0)
5.6 (0.630.4)
3.7 (0.418.0)
5.0 (0.028.1)
5.3 (0.222.6)
5.4 (0.627.8)
4.7 (0.411.1)
6.7 (0.328.1)
4.3 (0.223.5)
66.0 (0.090.0)
57.0 (4.0100.0)
40.5 (1.080.0)
39.0 (0.089.0)
3.4 (0.430.4)
6.5 (1.318.0)
3.2 (0.020.8)
6.5 (0.328.1)
3.1 (0.411.0)
5.8 (0.627.8)
4.1 (0.223.5)
7.8 (1.128.1)
68.8 (0.0100.0)
57.5 (2.01000)
41.0 (0.028.1)
40.3 (1.089.0)
6.3 (0.430.4)
3.7 (0.5174)
3.8 (0.028.1)
5.4 (0.3230)
4.9 (0.427.8)
4.6 (1.2148)
5.0 (0.228.1)
4.5 (0.3278)
66.0 (9.0100.0)
66.0 (32.096.0)
51.0 (0.092.0)
38.2 (4.092.0)
41.5 (2.087.0)
39.0 (10.081.0)
38.0 (1.083.0)
33.1 (0.089.0)
5.2 (0.630.4)
4.3 (0.421.1)
4.5 (2.217.1)
5.4 (2.96.5)
5.0 (1.023.0)
3.0 (0.020.8)
5.1 (0.728.1)
9.2 (6.322.6)
3.7 (0.627.8)
4.1 (0.411.4)
6.1 (1.511.1)
6.2 (5.611.0)
6.6 (0.328.1)
4.1 (0.223.5)
5.2 (1.114.4)
10.3 (6.412.8)
61.5 (2.0100.0)
57.5 (0.081.0)
70.4 (70.470.4)
39.5 (1.089.0)
35.0 (0.080.0)
64.2 (64.264.2)
4.0 (0.421.1)
7.0 (1.330.4)
6.4 (6.36.5)
4.7 (0.228.1)
7.6 (1.023.5)
12.8 (12.812.8)
5.4 (0.028.1)
2.9 (0.722.6)
7.4 (6.68.2)
5.4 (0.427.8)
1.9 (0.68.9)
9.6 (9.69.6)
HP5
HP2.5
HIF-1a
CAIX
Pimo-FC
Pimo
%
100
75.3
21.9
2.7
100
37.5
37.5
18.1
6.9
100
53.0
47.0
100
50.0
50.0
100
44.9
55.1
100
63.3
36.7
73
55
16
2
72
27
27
13
5
66
35
31
72
36
36
69
31
38
60
38
22
RESULTS
Patient characteristics and percentage of cells considered
hypoxic are given in Table 1. Frequencies of adenocarcinoma and squamous-cell carcinoma in this study reected the
incidence of these two types of cancer in the North American population (34,35). Similar ranges, means, and median
values for hypoxic fraction were found for pimonidazole,
CAIX, and HIF-1a staining. As in many previous studies
(3638), hypoxic fraction measured by three hypoxia
markers did not correlate with the well-established clinical
prognostic factors (including FIGO stage, maximum clinical
diameter, presenting hemoglobin, nodal status, and tumor
grade) (results of statistical analyses not shown).
Figures 1a1d illustrates the method used to determine
the percentage of tumor that was positive for each hypoxia
marker after antibody staining. Colored images were obtained by a x-y stage automatic tiling of entire sections
under 10 magnication (Fig. 1a). Tiled images were converted to gray scale images in Figure 1b. These underwent
thresholding to identify the entire area of the tumor (Fig. 1c)
as well as the marker positive region (Fig. 1d). Areas of
obvious necrosis or tissue folds were not included in the
analysis but no attempt was made to eliminate normal tissue components. Before comparing the staining patterns
for different hypoxia markers, it was important to establish whether immunohistochemical staining of sequential
sections was reproducible and consistent between sections. Fractions of the tumor stained for pimonidazole in
two sequential sections from 16 tumors were compared.
Results indicated a strong correlation (r 0.99, slope
1.1, data not shown).
In addition to immunohistochemical analysis, another
method was employed to measure pimonidazole binding.
A second biopsy was taken at the same time and within
30 min of biopsy, it was disaggregated with enzymes, and
xed in ethanol. Flow cytometry evaluation was performed with anti-pimonidazole antibodies. The percentage of hypoxic cells was calculated by tting ow histograms to three normal distributions representing aerobic,
intermediate, and hypoxic populations (29). For 30% of
the tumor samples, DNA content could be used to discriminate between diploid cells and hyperdiploid tumor
cells (Fig. 2). By gating on DNA content, hypoxic fraction
Histology
Squamous cell carcinoma
Adenocarcinoma
Adenosquamous
FIGO stage
I
II
III
IV
Grade
Well/mod differentiated
Poorly differentiated
Age (years)
<47 (median)
47 (median)
Size (largest diameter in cm)
<5.0 (median)
5.0 (median)
Nodal Status
Negative
Positive
Parameter
Statistical Analysis
Table 1
Clinicopathologic Parameters and Measures of Hypoxic Fraction. The Median Value of the Hypoxia Marker Positive Fraction and the Range of Values are Given
crotic regions (33) was not taken into account in measurement of degree of colocalization.
61.5 (2.096.0)
60.0 (0.0100.0)
49
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JANKOVIC ET AL.
FIG. 2. Representative ow cytometric analyses of pimonidazole binding in cells from cervical carcinoma biopsies. Single cells prepared from two
patient samples were xed and stained for pimonidazole adducts and for DNA content. The diploid are hyperdiploid cell populations are evident in panels (a) and (b), and the relation between DNA content and pimonidazole antibody binding is shown in panels (c) and (d). Panels in (e) indicate the distribution of pimonidazole antibody binding for diploid and hyperdiploid populations for each tumor, and the application of a curve tting algorithm to calculate hypoxic fraction. The hypoxic cell population (black) is on average 10 times more uorescent than the aerobic population (gray).
could be determined independently for tumor and normal cells. Invariably, the diploid population showed a
lower hypoxic fraction than the tumor cells, suggesting
that these cells are closer to functional blood vessels. A
reduced ability of diploid cells to metabolize and bind
pimonidazole was also observed, consistent with a
reduced nitroreductase activity. To compare hypoxic fraction measured by ow cytometry with hypoxic fraction
using image analysis of stained sections, DNA content
was not used to discriminate tumor cells from normal
cells even in those cases where this was feasible.
The percentage of hypoxic cells determined by this ow
cytometry approach agreed well with the much simpler
approach of visually scoring the fraction of brightly stained
cells cytospun onto slides after pimonidazole staining (Fig.
3a). Image analysis of pimonidazole-stained tumor sections
from a separate biopsy taken at the same time also showed
a signicant correlation with the ow cytometry method.
FIG. 3. Comparison of pimonidazole binding determined by ow cytometry, visual analysis of cytospun cells, or image analysis of stained
sections. Single cells prepared from biopsies were xed and labeled for
pimonidazole antibody binding. In panel (a), coded samples were analyzed by ow cytometry using the curve tting approach in Fig. 2 or by
visual counting of brightly stained cells after cytospinning. In panel (b),
results from the ow cytometry analysis of pimonidazole binding from
one biopsy were compared to the analysis of pimonidazole positive pixels from a second biopsy taken at the same time. Linear best-t lines
are drawn.
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JANKOVIC ET AL.
Table 2
Pearsons Correlation Coefcients Between Pairs of Hypoxia Measurement Techniques
Marker
statistics
Pimo
Pimo-FC
CAIX
HIF-1a
HP2.5
HP5
Pimo
Pearson
1
P
0
Pimo-FC
Pearson
P
0.45*
1
0
0
CAIX
Pearson
0.60*
0.36*
1
P
0
0
0
HIF-1a
Pearson
P
0.34*
0.13
0.42*
1
0
0.31
0
0
HP2.5
Pearson
0.12
0.07
0.03
0.02
1
P
0.91
0.59
0.83
0.91
0
HP5
Pearson
0.02
0.04
0.04
0.04
0.85*
1
P
0.89
0.76
0.79
0.75
0
0
Asterisk indicate that the correlation is signicant at the 0.01 level (2-tailed).
FIG. 5. Comparisons between hypoxia marker expression in cervical cancer biopsies. The percentage of
marker positive pixels are compared pair-wise in panels
(a)(c), and linear best-t lines are shown. Panels (d)
(f) present histograms of colocalization frequency between pairs of markers.
53
Table 3
Estimates of Marker Mismatch and Possible Explanations for Variation in Colocalization of Markers
Pattern observed
Frequency
(% of stained regions)
Pimo only
<5
HIF-1a only
10
CAIX only
10
1216
1417
57
All markers
6477
Note that under the category all markers, areas for comparison are dened based on a single marker
(e.g., do pimonidazole-stained regions express both HIF-1a and CAIX?) so regions of mismatch that do not
include that marker are ignored.
None of the three markers correlated with Eppendorf oxygen electrode measurement (median, HP 2.5 mmHg or
HP 5 mmHg). This is in spite of the fact that tumors with
high oxygen partial pressure (pO2) have been shown to exhibit decreased pimonidazole binding (40) and lower HIF1a expression (20). The relationship between marker expression and oxygen electrode measurements is a complex
one, as these methods do not sample the same tumor
microenvironment or provide directly comparable measures of hypoxia. There are now reports of a lack of correlation between oxygen electrode measurements and GLUT-1
expression (18), CAIX expression (15), and pimonidazole
binding (11).
Signicant correlations were observed between the
three single-cell measures of tumor hypoxia. Pimonidazole binding, HIF-1a, and CAIX expression all indicated
an average hypoxic fraction of about 6% in these tumors.
The mean/median values are similar to those previously
reported by Kaanders et al. for CAIX and pimonidazole in
head and neck cancers (6.4 and 6% respectively) (41),
and for pimonidazole binding in cervical cancers as reported by Azuma et al (4.5 6 4.8%) (42). They are higher
than the median value of 2% measured for HIF-1a in cervical cancers by Haugland et al (20). Other groups have used
a semi-quantitative scoring system making comparisons
more difcult. Underestimates of HIF-1a and CAIX staining
as a result of localization to nucleus or membrane, respectively, might be expected, and the dynamic range of staining
of tissue sections by the endogenous markers was typically
lower than for pimonidazole staining. However, the percentage of stained area is only one aspect of a comparison
between markers. Marker colocalization at a microregional
level was high in most cases, conrming that hypoxia is
likely to be the major factor in the expression of HIF-1a and
CAIX in these tumors. Lack of marker colocalization at a cellular level could be a useful indicator of the nature of tumor
hypoxia and at least some regions of some tumors showed
mismatch between these markers. Although HIF-1a stabilization under hypoxia is largely responsible for expression
FIG. 6. Comparison between HIF-1a and pimonidazole antibody staining patterns in sequential sections from three biopsies. Note that pimonidazole, unlike HIF-1a, appears to occur within necrotic regions in all three
of these tumors.
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JANKOVIC ET AL.
ACKNOWLEDGMENTS
This work was supported by the Canadian Institutes of
Health Research. The assistance of Genevieve Law during
a BCCRC summer studentship is gratefully acknowledged.
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