Vol.3, No.1, 6-12 (2011) Health
doi:10.4236/health.2011.31002
Copyright © 2011 SciRes. Openly acces sib le at http://www.scirp.org/journal/HEALTH/
Carbonic anhydrase VII–a potenti al pro gnostic marker in
gliomas
Fatemeh Bootorabi1,2, Joonas Haapasalo3, Elona Smith1, Hannu Haapasalo3,
Seppo Parkkila1,2
1Insti tute of Med ical Technology an d School of Medicin e, Univers ity of Tampere, Tampere, Fin land
2Department of Clinical Chemistry, Cent r e for Laborator y Med icine, Tampere University Hospit al, Tampere, Finland
3Department of Pathology, Centre for labor atory Medi cine, Tampere U niversity Hospital, Tampere, Finland;
*Cor responding Author :
Received 7 December 2010; revised 20 December 2010; accepted 26 December 2010
AB STRAC T
Carbonic anhydrase VII (CA VII) is a cytosolic
enzyme expressed in several organs, including
the human brain, but it has not been investi-
gated earlier in any tumors. We designed the
present study to ev alu at e C A VII exp r ession in a
cohort of human diffuse astrocytomas, mixed
oligoastrocytomas and oligodendrogliomas. CA
VII immunostaining was correlated to clinico-
pathologic findings, survival data, and expres-
sion of other molecular factors, including Ki-67,
p53 protein and epidermal growth factor recep-
tor. CA VII-positive staining was observed in
94% of astrocytomas and 85% of oligodendrog-
liomas. In the tumor specimens, strong positive
areas were often located in close proximity to
necrosis. The CA VII immunoreactivity showed
positive correlation with tumor malignancy grades
of astrocytomas (p = 0.02, chi-square test). In all
tumor categories, CA VII-positive staining was
often seen in the endothelial cells of neovessels
in addition to the tumor cells. CA VII intensity
showed no significant association with p53 nor
did it correlate with the amplification of epi-
dermal growth factor receptor (analyzed only in
astrocytomas) or cell proliferation. Our present
results show that CA VII may act as a useful
biomarker in histopathologic diagnostics of
gliomas. The high expression of CA VII in the
tumor cells and endothelium suggests impor-
tant roles for the enzyme in tumor metabolism.
The results also led us to conclude that CA VII
might serve as a marker of poor prognosis in
diffuse astrocytomas.
Keywords: Astrocytoma; Brain; Cancer;
Carbonic Anhydrase; Glioblastoma; Glioma;
Oligodendroglioma ; pH
1. INTRODUCTION
Carbonic Anhydrase (CAs) form a family of ubiquit-
ous zinc containing metalloenzymes that are able to cat-
alyze the reversible hydration of carbon dioxide accord-
ing to the following reaction: CO 2 + H2O ⇔ HCO 3− +
H+. The CAs show different distribution in a variety of
tissues where they participate in several important
biological processes such as pH regulation, CO2 and
HCO3− transport, respiration, ureagenesis, body fluid
generation, lipogenesis, and gluconeogenesis [1,2]. The
CA isozymes differ in several important characteristics,
such as kinetic properties, susceptibility to inhibitors,
and subcellular localization. The α-CA gene family has
been repo rted to include at le ast 13 active iso for ms with
d iff erent structural and catalytic properties [1-5]. CA
isozymes II, IX and XII have been extensively studied
for their important role as promising biomarkers in
different tumors, for example astrocytic tumors [5-7].
Other important research targets focused on CAs have
included hypoxia-regulation of certain CA isozymes and
their potential use as targets of cancer therapy [7-13].
Although cytosolic CA VII was identified as a new
CA isozyme almost twenty years ago [14], and its CA
catalytic activity was already demonstrated in 1996 [15],
the characteristics of this isozyme have largely remained
unresolved. In fact, the most developed research area on
CA VII has b een linke d to drug d evelop ment, which ha s
led to the discoveries of several potent activators and
inhibitors for this isozyme [16,17]. The expression of
CA VII protein was first studied in the b rain tissue [18 ].
In previous publications, CA VII has never been inves-
tigated in as troc ytomas nor has it been determined in any
other tumor categories. Data from GeneSapiens database
(http://ist.genesapiens.org/) suggested that some gliomas
express high levels of CA VII mRNA. This result

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7
prompted us to design the present study to evaluate the
expression of CA VII protein in diffusely infiltrating
astrocytic gliomas, oligodendrogliomas and mixed oli-
goastrocytomas. These tumors represent highly malig-
nant ne o p las ia s d e ri ved from gli al cell s. The y c o mmo nl y
show infiltrating and diffuse growth pattern, which
make s the s urgical treatment inadequate in most cases. It
is very important to diagnose these tumors as early as
possible and to use prognostic tools in the assessment of
the most suitable treatment for each patient. In the
present study, we evaluate the correlation of CA VII ex-
pression between astrocytomas, oligodendrogliomas and
oligoastrocytomas using immunohistochemistry and
compare the immunostaining results with various clini-
copathological and molecular factors including cell pro-
liferation, p53, and epidermal growth factor receptor
(EGFR).
2. METHODS
2.1. Study Material
The study material consisted of diffusely infiltrating
astrocytoma and oligodendroglioma samples, which
were obtained from surgically operated patients in Tam-
pere University Hospital, Tampere, Finland, during 1983
-2001. First, the tumor specimens were fixed in 4%
phosphate-buffered formaldehyde and processed into
paraffin blocks. On the basis of hematoxylin and eosin
(H&E)-stained slides a neuropathologist (HH) per-
formed an evaluation of the tumors according to the
WHO 2007 criteria [19,20]. According to the Finnish
legislation informed consent by the patients is not re-
quired in this kind of retrospective study. Most patients
had died before starting the analyses. According to the
national guidelines, the experiments were approved by
the Ethical Committee of the Tampere University Hos-
pital and the National Authority for Medicolegal Affairs
and conducted according to the guidelines of the Helsin-
ki Declaration.
2.2. Astrocytic Tumors
The WHO criteria divide diffusely infiltrating astro-
cytomas into three grades (II-IV) according to the pres-
ence of atypia, mitotic activity, necrosis and endothelial
proliferation. The neuropathologist selected one histo-
logically representative section in each sample specimen
for the CA VII immunohistochemistry. The study in-
cluded 107 astrocytic tumors (grade II: 14; grade III: 11;
grade IV: 82) and consisted of 90 primary tumors and 17
recurrences. Age of patients with primary tumors varied
from 20 to 80 years (median 55 ± SD 13 years) and re-
current tumors from 25 to 73 years (median 49 ± SD 14
years). Overall survival was kno wn for all of the patients
wit h p r imary tumors (90 patients; 12 grade II, 8 grade III
and 70 grade IV). The mean follow-up time for 14 sur-
vivors was 67 months (range 31-165) and 76 patients
died during the five-year follow-up. The tumors were
radically resected if possible and most patients with high
grade gliomas also received rad iotherapy.
2.3. Oligodendroglial Tumors
The WHO criteria group oligodendroglial tumors into
two main categor ies: pure oligodendroglio mas and mixed
oligoastrocytomas, which are divided into two grades (2
and 3) according to the at ypia, presence of increased cel-
lularity, and mitotic activity. From 47 cases of oligoden-
droglial tumor samples which were included in our ana-
lyses, there were both pure oligodendrogliomas (18 of
grade 2 and 12 of grade 3) and oligoastrocytomas (11 of
grade 2 and 6 of grade 3). These tumors included 35 pri-
mary tumors and 12 recurrences. The ages of the patients
with primary tumors varied from 8 to 76 years (mean ±
SD: 43 ± 14 years), and those of the patients with recur-
rent tumors varied fro m 17 to 72 year s (mean ± SD : 41 ±
14 years). Survival analysis was not performed, because
overa ll survi val was only known for 26 patients.
2.4. Immunohistochemistry
Five µm sections were processed for immunoperox-
idase staining, which was performed using an automated
Lab Vision Autostainer 480 (LabVision Corporation,
Fremont, CA, USA). Automated immunostaining was
performed using the Power Vision+ Poly-HRP Immuno-
histochemistry kit (ImmunoVision Technologies Co)
reagents. The primary rabbit anti-human CA VII serum
was raised against recombinant CA VII enzyme and has
shown high specificity for the CA VII enzyme [21]. The
immunostaining procedure included the following steps:
(1) rinsing in wash buffer; (2) treatment in 3% H2O2 in
ddH2O for five minute s and rinsing with wash b uffer; (3)
blocking with cow colostrum diluted 1:2 in Tris-buffered
saline (TBS) contai ning 0.05% Tween-20 for 30 minutes
and rinsing in wash buffer; (4) incubation with primary
antibody (rabbit anti-human CA VII for 30 minutes; (5)
rinsing in wash buffer three times for five minutes; (6)
incubation in poly-HRP-c onjugated anti-rabbit IgG for 30
minutes and rinsing in wash buffer three times for five
minutes; (7) incubation in DAB (3,3-diaminobenzidine
te trahyd rochlo ride) solution (one dr op of DAB so lution A
and one drop of DAB solution B in 1 ml of ddH2O) for
six minutes; (8) CuSO4 treatment for five minutes to en-
hance the signal; and (9) rinsing with ddH2O. All proce-
dures were performed at room temperature. The mount-
ing of the sections was performed using Entellan Neu
(Merck; Darmstadt, Germany). The intensity (INT) of
the staining was scored on a scale of 0 to 3 by three of

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the investigators (H. Haapasalo, F. Bootorabi and S.
Parkkila) as follows: 0, no reaction; 1, weak reaction; 2,
moderate reaction; and 3, strong reaction. The extent
(EXT) of staining was also scored on a scale of 0 to 3, 1
when 1-10% of the cells stained, 2 when 11-50% of the
cells stained and 3 when 51-100% of the cells stained. A
negative score (0) was given to tissue sections that had
no evidence of specific immunostaining. The relative
staining indices (SI) were calculated for each tumor
sample using the formula SQRT (EXT × INT) as de-
scribed earlier [22]. The tumors were then divided into
three groups based on the staining index: –, SI < 1 (im-
muno negat ive ); +, SI > 1 a nd < 2 (immunopositive); ++,
SI > 2 (strongly immunopositive).
In the present study, we correlated the expression of
CA VII to the extent of several other molecular markers,
such as Ki-67 (MIB-1, marker of cell proliferation) and
p53 as described previously [23]. EGFR amplification
was detected in astrocytic tumors using chromogenic in
situ hybridization (CISH) [24]. The immunostaining
results for CA IX and CA XII have been described earli-
er [7,25].
2.5. Statistical Analysis
All statistical analyses were performed using SPSS for
Windows (SPSS Inc. Chicago, IL). The significance of
associations was defined using chi-square test, Mann-
Whitney test, variance analysis and Kruskal-Wallis test.
The log ran k test, Kapla n -Meier c urves, a nd Co x multiva-
riate regression analysis were used in the survival analy-
sis. p-values < 0.05 were considered statistically signifi-
cant.
3. RESULTS
3.1. CA VII and Clinicopathologic Features
of Astrocytic Tumors
Cellular CA VII immunopositivity was observed in
101 cases of 107 (94%) diffusely infiltrating astrocyto-
mas. Strong immunostaining reactions (staining index
++) were observed in 47 specimens and weaker reac-
tions (staining index +) were present in 54 tumors. The
strongly positive areas were often located in close
proximity to necrotic regions (Figure 1). The positive
staining was usually unevenly distributed within the tu-
mor. It also appeared that the cell cytoplasm was more
inte nsive ly s taine d i n the tu mo rs with a napla sti c fe ature s.
In addition to the positive staining of tumor cells, CA
VII was ofte n located to t he endothelial ce lls of the neo-
vessels.
Within the group of WHO grade II astrocytomas 86%
of the tumors were CA VII-positive (50% + and 36%
Figure 1. Immunohistochemical staining of CA VII in repre-
sentative samples of glioblastomas (a-e) and oligodendroglioma
(f). Panels a-d show typical uneven distribution pattern of im-
munostaining. Stars in panels b and c show necrotic regions.
Strong immunoreactivity is often associated with hypo xic are as
locating in close proximity to necrosis. Panel e shows that posi-
tive signal did not follow the hypoxia-pattern in some cases.
Arrows indicate capillaries where endothelium is positive. In
the oligodendroglioma specimen, both the tumor cells and en-
dothelium (arrows) show positive immunostaining. Original
magnifications: × 200 (a,b), × 400 (c,e,f), × 630 (d).
++), and 82% of the grade III astrocytomas were CA
VII-positive (73% + and 9% ++) (Table 1). Interestingly,
almost all (98%) the grade IV tumors showed positive
immunostaining (48% + and 50% ++). The statistical
comparison of cytoplasmic CA VII staining index and
tumor grade revealed significantly higher CA VII stain-
ing in tumors with higher malignancy grade (p = 0.02,
chi-square test, Table 1). There was also a statistically
significant difference in staining indices between the
primary (44% + and 49% ++) and recurrent tumors
(82 % + and 18% ++), all of the recurrent tumors being
immunopositive (p = 0.015, chi-square test, Table 1).
The variance analysis showed that there was no signifi-
cant correlation between the CA VII staining index and
patient age (p > 0.05).
3.2. CA VI I and Molecular Pathologic
Features
When molecular pathologic features typical of astro

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Table 1. CA VII staining index (SI), WHO grade and recur-
rence status of the astrocytic and oliodendroglial tumors (p =
p-value, chi-squar e test).
CA V II imm unostaining
Tumor categor i es
Gra de
p
-
+
++
Total
Astroc ytom a
2
2
7
5
14
3
2
8
1
11
4
0.02
2
39
41
82
Oligodendrogliom a
2
3
9
6
18
3
>0 .05
2
2
8
12
Oligoastrocytoma
2
2
3
6
11
3
>0 .05
0
4
2
6
Primary tumor / Re-
currence
Astroc ytom a
Primary
6
40
44
90
Recurrenc e
0.005
0
14
3
17
Oligodendrogliom a
Primary
7
16
12
35
Recurrenc e
0.011
0
2
10
12
cytomas were compared with CA VII immunohistoche-
mistry, no association was observed between the cell
proliferation (assessed by Ki-67/MIB-1 immunostaining)
and CA VII expression (p > 0.05, Kruskal-Wallis test).
CA VII intensity showed no significant association with
p53 nor did it correlate with EGFR-amplification (p >
0.05, chi-square test). There was a significant correlation
between CA VII and CA IX staining index (p = 0.034,
chi-square test), whereas there was no significant rela-
tionship between CA VII and CA XII staining (p > 0.05,
chi-square test).
3.3. Correlation of CA VII with Prognosis in
Astrocytic Tumors
Overall survival data was known for all the patients
with primary tumors. Patient survival was tested by
log-rank test in relation to CA VII staining index. Inte-
restingly, CA VII stainin g results divided the tu mors into
three significantly different prognostic subsets (p =
0.017, log-rank test; Figure 2 ).
When different factors, includ ing CA VII, CA IX and
CA XII (all divided into three categories by staining in-
dices –, + and ++) as well as histological grade (2, 3 and
4) and patient age (<50, 50-65 and >65 years) were
tested in the Cox multivariate analysis, CA VII, CA IX,
histological grade and patient age were found to be of
independent prognostic value (Table 2).
3.4. Oligodendroglial Tumors
The study materials included 47 oligodendrogliomas of
which 30 cases represented pure and 17 were mixed oli
Figure 2. Survival of astrocytoma patients grouped
according to the CA VII immunostaining of tu-
mor cells. The patients with CA VII-negative tu-
mors had significantly better prognosis than those
with CA VII-positive tumors (p = 0.017, log-rank
test).
Table 2. The independent prognostic indicators of astro cytomas
as evaluated by Cox’s stepwise regression model. Histological
grade (2,3,4), patient age (cut points 50 and 65 years), CA VII,
and CA IX (staining index –, +, ++) were included in the
analysis.
Significance Exp(B)
(Hazard ratio)
95% CI of Exp(B)
Lower Upper
Grade 0.000 3.104 1.673 5.761
CA VII 0.002 2.074 1.306 3.293
Age 0.002 1.817 1.250 2.641
CA I X 0.046 1.431 1.006 2.034
goastrocytomas. 85% of all oligodendrogliomas showed
positive immunostaining for CA VII. There was no sig-
nificant difference in CA VII expression levels between
astrocytomas and oligodendrogliomas (p > 0.05, chi-
square test). Recurrent oligodendrogliomas were more
immunopositive for CA VII than the primary tumors,
and the difference was found to be statistically signifi-
cant (p = 0.011, chi-square test). There was no statisti-
cally significant correlation between the patient age and
CA VII status nor did CA VII correlate with tumor grade,
cell proliferation (assessed by MIB-1), or p53 immunos-
taining (p > 0.05, chi-square test). A near significant
correlation (p = 0.117, chi-square test) was observed
between CA VII and CA IX staining.
4. DISCUSSION
Previous studies have indicated that carbonic anhy-
drase isozymes, CA II , CA IX and CA XII, are promising
biomarkers for certain tumors [6,7,22,26]. There are nu-
merous reports showing that CA IX acts not only as a
marker for particular tumors, but its presence also corre-
lates to prognosis in several tumor categories, such as

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brain tumors, sarcomas, and renal, lung, rectal, bladder,
oral, breast, and cervical carcinomas [7,27-34]. Although
studies on CA II and CA XII have been mainly focused
on normal tissues, there are a few previous reports,
showing their presence in cancer cells and especially in
brain tumors where they have shown significant associa-
tion with prognosis [6,7,25]. CA VII is a cytosolic iso-
zyme which was first demonstrated in the brain tiss ue
[35], where it has been linked to the regulation of GA-
BAergic neuronal transmission in hippocampal neurons
[36]. We recently characterized this enzyme in vitro and
identified two forms of CA VII mRNA in the human
brain [21]. The studies further indicated that only the
full-lengt h form of CA VII is expres sed in human tissues.
The novel antibodies, raised against CA VII, demon-
strated that it is expressed in several other mouse tissues
-particularly in the liver. The present study was designed
when we found using the GeneSapiens database (http://
www.genesapiens.org), tha t CA7 gene is highly expressed
in some glioblastomas. The finding suggested that CA
VII could represent another CA isozyme with a potential
rol e as a biomarker for gliomas.
In this study, we investigated the expression of CA
VII in three different categories of brain tumors: astro-
cytomas, oligodendrogliomas and mixed oligoastrocy-
tomas. Our findings showed that CA VII expression is
higher among high grade infiltrating tumors. Interes-
tingl y, CA VII immuno staining was often ver y strong in
hypoxic areas adjacent to tumor necrosis. Although CA
VII has not been considered a HIF-regulated isozyme
contrary to CA IX [37,38] and CA XII [38], its staining
pattern indicates that ce ll hypoxia co ntributes to the reg-
ulation of CA VII expression by yet undefined mechan-
isms. According to our results CA VII immunoreactivity
did not correlate significantly with cell proliferation de-
termined by MIB-1 immunostaining, nor did it show
association to p53 protein expression or EGFR- amplifi-
cation. Importantly, our results indicated that high CA
VII expression is associated with poor prognosis in as-
trocytoma patients. This result makes CA VII another
C A iso zyme with a significant correlation to the patient
survi val. I ndeed , the p revio us stud ies ha ve de monstrated
that CA II, CA IX a nd CA X II all show similar trends as
prognostic markers in diffuse astrocytomas [6,7,25,39].
The presence of several CA isozymes in astrocytomas
may reflect the rapid turnover of acid metabolic products
in highly malignant tumor tissues. The cytosolic isozymes,
such as CA II and CA VII, can contribute to more effi-
cient neutralization of cell interior, whereas the mem-
brane-associated enzymes, CA IX and CA XII, partici-
pate in extrusion of protons in metabolon systems to-
gether with ion transport proteins [5,40,41]. These me-
chanisms can provide novel opportunities for cancer
therapy in which tumor cell microenvironment can be
targeted by CA inhibition. Recent drug developments
have already pointed out a number of different com-
pounds as CA inhibitors with high efficiency [5,40].
Some of the drugs may inhibit and disturb the neoangi-
ogenesis while reducing the tumor growth [42], and
some compounds can also decrease the invasion capacity
of tumor cells [43,44].
In conclusion, our findings demonstrate that CA VII
isozyme is highly expressed in several cases of malignant
brain tumors including oligodendrogliomas, oligoastro-
cytomas and diffusively infiltrating astrocytomas. The
positive immunostaining correlates with poor prognosis
of patients with astrocytomas. The presence of several
CA isozymes in malignant brain tumors may provide
novel opportunities for developing cancer treatment
strategies targeted to the microenvironment of cancer
cells.
5. ACKNOWLEDGEMENTS
The authors thank Aulikki Lehmus and Reija Randen for skilful
technical assistance. This study was funded by European Union DeZ-
nIT project, Academy of Finland, Sigrid Juselius Foundation, and
Competitive Research Funding of the Tampere University Hospital
(9L071).
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