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Regular Articles |
From the Institute of Molecular Medicine*
and the
Breast Screening Program,§
John Radcliffe
Hospital, Oxford, United Kingdom; the Wellcome Trust Centre for Human
Genetics,**
Oxford, United Kingdom; the
Department of Pathology,
University of
Manitoba, Winnipeg, Manitoba, Canada; the Division of Medical
Oncology,
British Columbia Cancer Agency,
Vancouver, British Columbia, Canada; the Institute of
Virology,¶
Slovak Academy of Sciences, Bratislava,
Slovak Republic; and the Edward A. Doisy Department of
Biochemistry,||
St. Louis University School of Medicine,
St. Louis, Missouri
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Necrosis is believed to represent the extreme manifestation of hypoxia within tissues.11 Interestingly, tumor hypoxia has been shown to be a prognostic indicator for many tumor types, being associated with aggressive growth, metastasis, and poor response to treatment not only in patients treated with radiotherapy, but also in those treated with surgery alone.12-16 Of potential importance for understanding these effects is the role of hypoxia in regulating patterns of gene expression. Studies of gene expression have defined several classes of genes that are up-regulated by hypoxia and demonstrated that activation of the transcriptional complex hypoxia-inducible factor-1 is a key mediator of many of these effects.17,18 Genes that are up-regulated by microenvironmental hypoxia through hypoxia-inducible factor-1 activation include glucose transporters, glycolytic enzymes, and angiogenic growth factors.
We recently identified the two tumor-associated transmembrane carbonic anhydrases (CA) CA919-21 and CA1220,22 as being up-regulated by hypoxia in epithelial tumor cell lines.23 Furthermore, we demonstrated focal perinecrotic expression of CA IX in invasive human tumors, co-localizing with vascular endothelial growth factor mRNA expression and pimonidazole activation.23
CA9 and CA12 are members of a family of catalytically active CAs that share the capacity to catalyze the reversible hydration of carbon dioxide to carbonic acid.24 CA IX19,21,25 has been studied extensively in several tumor types including lung, kidney, colon, and cervix, where its expression has been established as a marker of cellular proliferation and early dysplasia.26-30 CA XII was initially identified in renal carcinoma,22 and subsequently shown to be associated with colon carcinoma where altered expression occurs in early stages of tumorigenesis.31 However, the expression of these CAs in breast cancer has not been examined.
We investigated CA IX and CA XII expression in breast cancer in anticipation that their expression might serve as indicators of tissue hypoxia, altered pH, and tumor progression. Specifically we wished to assess the pattern of expression of these genes in DCIS, where the appearance of necrosis and abnormal calcification is associated with a high risk of progression to invasive disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sixty-eight pathological specimens containing DCIS of the breast were selected from review of surgical resections collected from 1997 to 1999 at the John Radcliffe Hospital and the Churchill Hospital, Oxford, UK. The cohort comprised 39 cases of pure DCIS (DCIS-) and 29 cases of DCIS associated with invasive carcinoma in the same biopsy (DCIS+), either independent from or directly associated with adjoining invasive carcinoma.
All DCIS lesions were classified into histological grades on the basis of the predominant grade present in the tissue section studied for gene expression according to the Van Nuys grading system.9,32 The presence of intraductal necrosis within any component of DCIS within the tissue section was evaluated in hematoxylin and eosin-stained sections by light microscopy. The radiological appearance was classified according to the presence and pattern of calcification5,6 in preoperative mammograms for a subset of cases in which films were available (n = 43). These classifications were performed by a single pathologist (PHW) and radiologist (RE), respectively, without reference to the cohort’s immunohistochemical data and outcome. Among the series of cases, the histological grades were as follows: 18 low grade (8 DCIS-, 10 DCIS+), 24 intermediate grade (15 DCIS-, 9 DCIS+), and 26 high grade (16 DCIS-, 10 DCIS+). Intraductal necrosis was present in 51 cases (75%), among which 29 were DCIS- and 22 DCIS+. Mammographic calcifications were present in 35 of 43 cases, among which 27 of 35 were DCIS- and eight of 35 DCIS+. The pattern of calcification was classified as linear type (14 cases) if the presence of any linear calcification was seen, nonlinear type (21 cases), or absent (eight cases).
Cell Lines and Immunoblotting
MDA-MB-231 and ZR-75.1 cell lines were from ATCC (Rockville, MD). Hypoxic conditions were generated in a Napco 7001 incubator (Precision Scientific, Winchester, VA) with 0.1% O2, 5% CO2, and balance N2 for 16 hours. Whole-cell protein extracts were prepared from tissue culture cells by 10 seconds of homogenization in denaturing conditions as described.33 Whole-cell protein extracts were prepared from tumors by fine section of frozen tissue and 30 seconds of homogenization in denaturing conditions identical to tissue-culture extracts. For Western analysis, aliquots were separated under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, Hertfordshire, UK). CA IX was detected using the mouse monoclonal anti-human CA IX antibody M75 (1:50) as described.34 CA XII was detected using a rabbit polyclonal anti-human CA XII antibody (1:2000) as described.22 horseradish peroxidase-conjugated goat-anti-mouse and swine anti-rabbit immunoglobulins (DAKO, Cambridgeshire, UK) (1:2000), respectively, were applied for 1 hour at room temperature. ECL Plus (Amersham Pharmacia, Buckinghamshire, UK) was used for visualization.
Immunohistochemistry (IHC)
Formalin-fixed, paraffin-embedded tissue specimens collected by standard surgical oncology procedures were obtained from the Pathology Department, John Radcliffe Hospital, Oxford, UK. Immunostaining of paraffin sections was performed after dewaxing and rehydrating 5-µm sections. Staining for CA IX, CA XII, and MIB1 was performed on serial sections. Endogenous peroxidase was blocked with 0.5% hydrogen peroxide in water for 30 minutes. Ten percent normal human serum in Tris-buffered saline was applied for 15 minutes at room temperature to block nonspecific protein binding. Primary antibodies: anti-human CA IX murine monoclonal antibody M75 (1:50);35 anti-human CA XII rabbit polyclonal antibody (1:2000); anti-human Ki67 murine monoclonal antibody MIB1 (1:50) (Immunotech). Primary antibodies were incubated for 30 minutes at room temperature in Tris-buffered saline with 5% normal human serum, followed by a 30-minute incubation with a peroxidase-conjugated secondary antibody. After each incubation, slides were washed twice with Tris-buffered saline for 5 minutes. Visualization of staining was by diaminobenzidine substrate for 8 minutes. Slides were counterstained with hematoxylin before mounting in Aquamount (BDH). Substitution of primary antibody with PBS was used as a negative control for each antibody. All staining was performed on an automated IHC stainer (MiniPrep 75; Tecan) at room temperature.
Assessment of CA IX, CA XII, and MIB1 Staining
Immunostaining for CA IX and CA XII was assessed by light microscopy and semiquantitative scoring was performed by a single pathologist (PHW), independently of the pathological assessment. Expression and intensity was scored (0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining), together with the percentage of normal or neoplastic epithelial cells showing expression within the tissue section (0 to 100%). The product of the intensity and the percentage gave a final immunostaining score (0 to 300; IHC score). MIB1 expression was assessed using a Chalkley point array method adapted from methods used to assess vascular density in breast sections.11 Briefly, MIB1-immunostained section was reviewed at low magnification and five areas showing the highest density of MIB1-positive tumor cells were selected. These hot spots were then assessed at higher magnification (x25 objective) and the number of grid points that coincided with positive and negative tumor cell nuclei was counted. The mean ratio of MIB1-positive/MIB1-negative cells was then calculated. Each hot spot contained between 200 and 1000 tumor cells depending on DCIS histology.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-CA IX and anti-CA XII antibodies used in this study have
been previously characterized for immunostaining in many human
tissues.22,31,34,36
However, neither antibody has been
applied extensively to breast specimens. Therefore, initial experiments
were performed to compare IHC profiles with immunoblotting signals from
a set of six invasive breast ductal carcinomas. By IHC, two cases
exhibited strong membranous staining for CA IX that was restricted to
the invasive ductal carcinoma cells, one was weakly positive, and three
were negative (data not shown). Two of the tumors that were either
negative or weakly positive for CA IX by IHC exhibited strong
membranous staining for CA XII, whereas four cases were negative (data
not shown). Immunoblotting for CA IX and CA XII was performed in
parallel on protein lysates obtained from the same tumor specimens. As
shown in Figure 1A
, immunoblotting for CA
IX revealed a 54- to 58-kd doublet restricted to the two cases that
were strongly positive by IHC. Similarly, immunoblotting for CA XII
revealed a 46- to 48-kd doublet restricted to the two cases that were
positive by IHC.
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We have previously demonstrated wide-spread hypoxic induction of
CA9 and CA12 mRNA in various tumor cell
lines.23
Here we compared hypoxic induction of CA IX and
CA XII in two breast cell lines selected as representative of estrogen
receptor (ER)-negative and poorly differentiated (MDA-MB-231), and
ER-positive and well-differentiated (ZR-75.1) breast cancer (Figure 1B)
. CA IX had an undetectable or low basal level of expression and was
markedly induced by hypoxia. In comparison, CA XII had a higher level
of normoxic expression and was further induced by hypoxia in one of the
two cell lines.
CA IX Expression in Breast Tissues
A series of 68 DCIS breast cases were studied for CA IX expression
by IHC. Subsets of these cases also contained normal lobular and ductal
components (n = 47), and invasive ductal
carcinoma components (n = 29). CA IX expression
was present in normal epithelium in one of 47 cases (2%), and in this
case was limited to focal expression adjacent to the site of a recent
biopsy. In many cases, benign breast lesions were present within the
tissue section, including cystic changes, apocrine metaplasia, blunt
duct, and sclerosis adenosis. No expression was observed in any benign
breast lesion. In DCIS lesions, focal membranous CA IX staining,
typically adjacent to areas of necrosis, was present in 34 of 68 (50%)
cases, including 23 of 39 (59%) pure DCIS and 11 of 29 (38%) DCIS
associated with invasive disease. In those cases in which invasive
disease was present on the same tumor section, CA IX was expressed
adjacent to regions of necrosis where this was present within the
invasive component in four of 14 cases (29%). The presence of CA IX
staining in both DCIS and invasive components was correlated
(r = 0.55, P = 0.04). The focal
perinecrotic nature of expression was reflected in the distribution of
IHC scores with only 13 (19%) tumors scoring >10 (potential range of
IHC score was 0 to 300, as described in Materials and Methods). The
range of IHC scores was from 0 to 100 (median, 1; mean, 9; and SD, 17).
Representative examples of low and high CA IX expression are
illustrated in Figure 2, A and B
. CA IX
was significantly associated with high grade (grade low
versus intermediate versus high; mean (SD), 2
(5), 11 (16), 13 (22), P = 0.012 analysis of variance)
and the presence of necrosis (necrosis negative versus
positive; mean (SD), 2 (5), 12 (19), P = 0.0053,
Mann Whitney; Figure 3
and Table 1
).
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The expression of CA XII was assessed by IHC in sections adjacent
to CA IX-stained sections for all 68 DCIS cases. Membranous staining of
the basal-lateral aspects of breast epithelial cells was present in
normal lobular and normal ductal epithelium in 42 of 47 (89%) cases
and in every benign breast lesion observed (Figure 2, C and D)
. In
ductal hyperplasia, CA XII expression was predominately limited to
basal epithelial cells. In DCIS lesions, widespread membranous CA XII
staining was present in 57 of 68 (84%) cases, including 31 of 39
(79%) pure DCIS and 26 of 29 (90%) DCIS associated with invasive
disease. In those cases in which invasive disease was present on the
same tumor section, CA XII was expressed in 10 of 14 cases (71%). The
presence of CA XII staining in both DCIS and invasive components was
highly correlated (r = 0.91, P
< 0.0001). Although expression in DCIS was typically homogeneous
throughout the tumor section, in intermediate- and high-grade DCIS
where CA XII expression tended to be lower, expression was increased
adjacent to areas of necrosis (Figure 2E)
. The distribution of IHC
scores was wider than for CA IX, with 17 (25%) tumors scoring 60 or
more (potential range, 0 to 300). The range of IHC scores was from 0 to
270 (median, 40; mean, 54; and SD, 61). Representative examples of low
and high CA XII expression are illustrated in Figure 2, E and F
. CA XII
was significantly associated with low grade (grade low
versus intermediate versus high; mean (SD),
79(57), 50(69), 40(52), P = 0.012 analysis of variance)
and the absence of necrosis (necrosis negative versus
positive; mean (SD), 68(52), 49(64), P = 0.036, Mann
Whitney; Figure 3
and Table 1
).
CA IX and CA XII Expression Relative to the Proliferation Marker MIB1
A correlation between CA IX and proliferation has been suggested
previously.26
We therefore examined the relationship
between proliferation and CA IX and CA XII in our breast tissue
specimens (Figure 4)
. Comparison of
mitotic rates within positively stained ducts for CA IX and CA XII and
within different zones in these ducts (adjacent to the stroma or the
lumen), indicated that neither CA IX nor CA XII expression correlated
regionally with mitosis. Whereas CA IX expression was typically
localized to areas adjacent to necrosis, mitotic figures did not show a
similar distribution, being most numerous in the cells adjacent to
stroma and farthest removed from necrosis. Similarly, CA XII expression
was not restricted to the areas of highest mitotic activity and was
typically uniform throughout the intraductal epithelium, with
occasional accentuation in luminal cells adjacent to necrosis. To
confirm these morphological observations, a random subset of cases
(n = 26) were immunostained for the
proliferation marker MIB1 and the MIB1 score was determined by Chalkley
counting. In agreement with previous observations,37
we
found MIB1 to be associated with both grade (low versus
intermediate versus high; mean (SD), 0.16(0.07), 0.4(0.18),
0.56(0.33), P = 0.0026 analysis of variance) and the
presence of necrosis (necrosis negative versus positive;
mean (SD), 0.19(0.09), 0.51(0.28), P = 0.001 Mann
Whitney; Figure 5
). However, MIB1 was not
significantly related to the expression of either CA IX or CA XII (CA
IX negative versus positive; MIB1 mean (SD), 0.44(0.37),
0.37(0.21); CA XII negative versus positive; MIB1 mean (SD),
0.43(0.31), 0.38(0.26); Table 1
). Similarly, when CA IX and CA XII
expression were divided into low and high expression using the median
IHC score of the series for each (CA IX positive, >1 and CA XII
negative, >40), no association was detected between the expression of
either CA and MIB1 staining.
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CA IX and CA XII expression were assessed in relation to the
presence and pattern of calcification detected in preoperative
mammograms in a subset of cases in which films were available for
review (n = 43). The presence of calcification
was associated with the presence of necrosis (P
= 0.0036, chi-square) and higher grade (P =
0.0057, chi-square) (Table 2)
. When CA
gene expression was classified as low or high on the basis of the
median IHC score of the series (CA IX positive, >1 and CA XII
positive, >40) a significant relationship was observed between lower
CA XII expression and the presence of calcification
(P = 0.0083, chi-square), as shown in Table 2
.
The level of CA XII expression was also inversely associated with
calcification (calcification absent versus present, mean
(SD), 94(60), 42(60), P = 0.03, Mann Whitney). The
pattern of calcification was not significantly different with respect
to either CA gene expression. Despite this, comparison of cases with
nonlinear versus cases with some component of linear
calcification revealed a trend toward an increased proportion of CA IX
positive cases (8 of 21 vs. 9 of 14 or 38% vs.
64%), whereas the proportion of CA XII positive cases was no different
(6 of 21 vs. 3 of 14 or 29% vs. 21%).
Additionally, cases with linear calcification tended to be associated
with higher levels of CA IX expression than cases with nonlinear
calcification (CA IX IHC score mean (SD), 9 (12) vs. 5 (10),
P = n.s.) whereas there was no such trend for CA XII
(CA XII IHC score, 41 (69) vs. 43 (55), P =
n.s.).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CAs have been studied in a spectrum of tumor types in relation to their potential role as diagnostic and prognostic markers. Earlier studies focusing on CAs I, II, and IV revealed no clear relationships with tumorigenesis.26 More recently, CA9 has been identified as overexpressed in multiple tumor cell lines and in several human tumor types. In various studies, CA9 has been found to be associated with aspects of early tumorigenesis,20,26,30,35,38 and it has therefore been proposed to serve as a biomarker for dysplasia. In accordance with these findings, we have shown that whereas CA IX expression is rare in normal or benign breast lesions, CA IX expression occurs in pre-invasive DCIS of the breast where it is limited to malignant epithelium. In the breast specimens examined, CA IX expression was not related to proliferation and was strongly associated with necrosis, indicating that hypoxia may be an important pathway for induction of CA IX in breast tumors in vivo.
CA12 was initially identified as a renal
carcinoma-associated gene20,22
and has subsequently been
found to be expressed in a range of normal tissues including
endometrium, pancreas, and colon.31,39,40
Interestingly,
in the normal colon CA XII is expressed highly by the differentiated
surface epithelium relative to the cells of the crypt base, and whereas
no change in the surface expression occurs with tumorigenesis,
increased basal/deep mucosal expression is associated with increasing
dysplasia and invasive tumor stage.31
Similarly, and in
striking contrast to CA IX expression, we have observed constitutive
expression of CA XII in normal breast epithelium and benign ductal
hyperplasia. This suggests that CA XII may play a role in the control
of pH in normal breast tissue. The function of this membrane-associated
extracellular CA may be coupled to that of an intracellular CA such as
CA II, as has been hypothesized for other secretory/excretory organs
such as the salivary glands, pancreas, and kidney.40
Clearly, a detailed examination of the interplay between the many CAs
is warranted. We have also shown that CA XII expression persists in
malignant pre-invasive DCIS. Although focal induction of CA XII was
observed in areas adjacent to necrosis, the differentiation status of
the DCIS lesion (as indicated by grade) had a more dominant role in
determining CA XII expression, which was reflected in the pattern of
expression observed in the ER-negative and ER-positive cell lines
examined. Of note, differentiation has been proposed to play a role in
the expression of other CAs, including CA I whose induction is
association with differentiation in the colon,41
and CA II
whose expression is associated with differentiation in pancreatic cell
lines under the influence of tumor necrosis factor-
.42
An abnormal pattern of calcification in a breast mammogram is an important indicator of DCIS.5 In particular, the presence of linear type calcification is associated with high-grade DCIS and may predict outcome of associated small invasive tumors.5,6,43,44 Calcification is believed to reflect a disruption of the normal vascular architecture caused by abnormal proliferation within the intraductal epithelium.45 This leads to a reduction in luminal pH, changes in the equilibria of many ions, and resulting calcification.46 Inherited alteration and deficiency of CA II activity causes metabolic acidosis and ectopic tissue calcification.47 Similarly, changes in extracellular pH influenced by CA IX and CA XII expression may affect the extent and pattern of calcification in DCIS of the breast. In the current series, increased mammographically detectable calcification was associated with reduced CA XII expression, as well as the presence of high-grade DCIS and necrosis, as previously reported.48 Our inability to demonstrate a relationship between calcification and CA IX staining could relate to the fact that CA IX staining was only present very focally. Therefore, the tissue block assessed for CA IX expression may not correspond to the status of the area of the mammogram assessed for calcification, which encompassed the entire biopsy. Furthermore, our results suggest that whereas overall loss of CA XII expression is important in the development of calcification, local gain of CA IX expression adjacent to the ductal lumen may influence the pattern of calcification. However the significance of these observations awaits confirmation by larger studies as this subset of cases is small and includes a disproportionate number of cases with calcification present, reflecting the fact that calcification is a key factor in detection of tumors by mammography.5
The effect on local pH and the significance for breast tumor progression of reciprocal changes in the expression of these CAs remains to be determined. However, there is additional evidence to suggest that a switch in pH regulatory pathways may occur in breast tumor progression. Although a decrease in the activity of the Na+/H+ exchanger was noted in response to serum deprivation in nontumor breast cells, stimulation of this exchanger and an increased capacity for extracellular acidification was observed in tumor cells.49 In terms of tumor progression, maintenance of high levels of CA XII may be important for both the function and survival of the ductal epithelium in normal tissue. Loss of CA XII expression with progression to higher grade DCIS may reflect the acquisition of alternative cellular responses to ameliorate the effects of disruption of tissue architecture, altered pH, and hypoxia.18,50 One facet of this adaptation may be provided by the induction of other CAs such as CA IX to modulate the effects of local hypoxia. This view predicts that overall loss of CA XII and/or gain of CA IX expression may be associated with a high risk of progression to invasive disease and therefore be of prognostic significance. Interestingly, inhibition of CA activity has recently been demonstrated to suppress invasion of some tumor cell lines.51
In conclusion, we have shown that CA IX and CA XII are expressed in breast tissues, and that the profile of expression of these CAs in DCIS suggests that whereas hypoxia may be a dominant factor in the regulation of CA IX, the regulation of CA XII is dominated by other factors related to cellular differentiation.
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Footnotes |
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Supported by the Imperial Cancer Research Fund and the Wellcome Trust. P. H. W. is supported by a Scientist Award from the Medical Research Council of Canada, an Academic Award from the U. S. Army Medical Research and Materiel Command, and a Research Travel Fellowship from Burroughs Wellcome. S. K. C. is supported by the Shane Fellowship and the Canadian Breast Cancer Foundation.
Accepted for publication December 4, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. K. Chia, C. C. Wykoff, P. H. Watson, C. Han, R. D. Leek, J. Pastorek, K. C. Gatter, P. Ratcliffe, and A. L. Harris Prognostic Significance of a Novel Hypoxia-Regulated Marker, Carbonic Anhydrase IX, in Invasive Breast Carcinoma J. Clin. Oncol., August 15, 2001; 19(16): 3660 - 3668. [Abstract] |
