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© 2001 American Society for Clinical Oncology Prognostic Significance of a Novel Hypoxia-Regulated Marker, Carbonic Anhydrase IX, in Invasive Breast CarcinomaByFrom the Division of Medical Oncology, British Columbia Cancer Agency, Vancouver, British Columbia; Department of Pathology, University of Manitoba, Winnipeg, Manitoba, Canada; Imperial Cancer Research Fund Molecular Oncology Laboratory, University of Oxford, Institute of Molecular Medicine and Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, and Welcome Trust, University of Oxford, Churchill Hospital, Oxford, United Kingdom; and Institute of Virology, Slovak Academy of Sciences, Slovak Republic. Address reprint requests to Adrian L. Harris, MD, Imperial Cancer Research Fund Molecular Oncology Laboratory, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom; email: aharris.lab{at}lcrf.lcnet.uk
PURPOSE: To assess the frequency of expression and the prognostic significance of a hypoxia-regulated marker, carbonic anhydrase IX (CA IX), in a cohort of patients with invasive breast cancer. PATIENTS AND METHODS: CA IX expression was evaluated by immunohistochemistry with a murine monoclonal antibody, M75, in a series of 103 women treated surgically for invasive breast cancer. The majority of patients were treated with adjuvant hormonal or chemotherapy. The frequency of CA IX expression, its association with recognized prognostic factors, and the relationship with outcome was evaluated by univariate and multivariate statistical analyses. RESULTS: CA IX expression was present in 49 (48%) of 103 cases. The level of CA IX expression was found to be significantly associated with tumor necrosis (P < .001), higher grade (P = .02), and negative estrogen receptor status (P < .001). Furthermore, CA IX expression was associated with a higher relapse rate (P = .004) and a worse overall survival (P = .001). By multivariate analysis, CA IX was also shown to be an independent predictive factor for overall survival (hazard ratio, 2.61; 95% confidence interval, 1.01 to 6.75, P = .05). CONCLUSION: CA IX expression was associated with worse relapse-free survival and overall survival in an unselected cohort of patients with invasive breast carcinoma. The potential role of CA IX as a marker of hypoxia within breast carcinomas was also indicated by a significant association with necrosis. Further work assessing its prognostic significance in breast cancer is warranted, particularly interactions with radiotherapy and chemotherapy resistance.
THE BENEFITS OF adjuvant chemotherapy and hormonal therapy for the treatment of invasive breast cancer are now well proven.1,2 However, many women still undergo treatment without necessarily achieving benefit. Much work has been directed at identifying prognostic factors that will improve our capability to predict the risk of breast cancer relapse and death in women after the diagnosis of primary breast cancer. The value of factors such as axillary lymph node status, tumor size, tumor grade, and hormonal receptor status are well established.3-6 However, earlier diagnosis and changes in clinical practice have made it more difficult to apply all of these factors because the majority of women now present with small, node-negative tumors. Further work is required to refine relapse risk in order to determine the group of women most in need of adjuvant treatment and to spare those whose prognosis is favorable further therapy. Also, it is important to identify factors that may modify the effectiveness of therapy because these may provide new targets for modification of resistance. Hypoxia has been implicated as an important component in tumor progression and spread. The degree of tumor hypoxia has been shown to be inversely correlated with response to treatment and overall survival.7 This is partly related to radiation resistance but is also independently a risk factor for poor outcome.8,9 Hypoxia is also a vital factor in the etiology of tumor necrosis, and the latter parameter has also been demonstrated, although perhaps not widely appreciated, to be a prognostic factor for a worse relapse-free and overall mortality rate in both node-negative and node-positive breast cancer.10 A correlation has been established between the degree of tumor necrosis and angiogenesis.11,12 Last, the degree of angiogenesis, as quantified by microvessel density and analysis of angiogenic factors such as vascular endothelial growth factor in tumor tissue samples, has been found to be prognostic for relapse-free survival (RFS) and overall survival (OS) in cohorts of women with node-positive and node-negative breast carcinoma.13-18 Taken together, it may be plausible that hypoxia, tumor necrosis, and angiogenesis are potentially associated biologically and prognostically in breast carcinoma. We have recently identified the carbonic anhydrase 9 (CA 9) gene to be hypoxia-inducible in several epithelial cell lines via a hypoxia response element in its 5' promoter. Furthermore, the upregulation of this enzyme was dependent on hypoxia inducible factor 1.19 Carbonic anhydrase 9 protein (CA IX) belongs to the family of zinc metalloenzymes responsible for the reversible conversion of carbon dioxide and water to carbonic acid. Members of the mammalian alpha-CA family play a variety of roles in many physiologic processes, in particular serving to maintain pH, water, and ion equilibrium.20,21 CA IX, originally identified in HeLa cells (a human cervical carcinoma cell line), was also found to be a tumor marker in human ovarian, endometrium, and cervical cancer specimens, and was named MN.22 Subsequently, the MN cDNA was cloned and the transmembrane protein product showed structural and functional homology with other alpha-CA isoenzymes23,24 and was subsequently renamed CA 9.25 CA IX is normally expressed in the alimentary tract and associated organs.26,27 Further work has shown that CA IX is expressed in renal cell carcinoma,28,29 esophageal carcinoma,30 non–small-cell lung carcinoma,31 and colorectal tumors.32 These studies have also revealed that the expression of this biomarker is restricted to transformed, dysplastic, and malignant epithelial cells and is rarely expressed in benign tumors or normal tissue. To date, we are unaware of any extensive reports investigating the presence of CA IX in human breast cancer or on the relationship of CA IX expression to outcome in any human cancer series. Therefore, in light of our recent findings that CA IX may be a marker for hypoxia in vivo, the aims of this study were to assess the expression of CA IX in invasive breast carcinoma in relation to tumor necrosis and to determine whether this novel biomarker is an independent prognostic factor for breast cancer relapse and death.
Patients and Tissues We assessed a series of 103 surgically resected available invasive breast carcinomas treated at the John Radcliffe Hospital and the Churchill Hospital, Oxford, from 1989 to 1994 with a median follow-up of 6.2 years. The majority of the cohort underwent either modified radical mastectomy or lumpectomy with breast irradiation. Axillary lymph node status was confirmed histologically. If lymph node involvement was found, then adjuvant radiotherapy to the axilla was delivered. Adjuvant systemic treatment consisted of tamoxifen at 20 mg daily for 5 years for postmenopausal women regardless of hormonal receptor status and six cycles of intravenously administered cyclophosphamide, methotrexate, and fluorouracil delivered every 3 weeks for premenopausal women. All patients were assessed at follow-up every 3 months for the first 18 months and every 6 months thereafter. Treatment for confirmed recurrent disease was by endocrine manipulation for soft tissue or skeletal metastasis or by chemotherapy for visceral disease and failed endocrine therapy. The clinicopathologic characteristics of the entire cohort are summarized in Table 1.
Immunohistochemistry Immunohistochemical staining for CA IX was performed on 5-µm serial sections on coated slides from paraffin-embedded blocks. Paraffin was first removed from all slides by means of standard techniques; slides were then placed in 0.5% hydrogen peroxide for 15 minutes to saturate endogenous peroxidases. Incubation with 10% normal human serum in Tris-buffered saline (TBS) for 15 minutes was then performed to block nonspecific uptake of the antibody. The murine monoclonal antibody M75 at a dilution of 1:50 in TBS with 5% normal human serum for 30 minutes was used to assess expression of CA IX. This antibody has previously been characterized with Western blot analysis to confirm its specificity.27 We have also further confirmed its ability to specifically detect CA IX expression in tissue sections by direct correlation with Western blot analysis in human breast tumor specimens.33 Known positive and negative controls for CA IX expression from this direct correlation were then used with each immunohistochemistry (IHC) procedure batch to ensure quality control of staining. Next, a 30-minute incubation with a peroxidase conjugated to goat anti-mouse immunoglobulins (Dako EnVision + System, peroxidase, mouse; Dako, Carpinteria, CA) was performed. Slides were then stained with 3,3'-diaminobenzidine chromogen solution for 8 minutes, then counterstained with hematoxylin and mounted with Aquamount. All staining was performed on an automated immunohistochemical stainer (Miniprep 75; Tecan, Reading, United Kingdom) at room temperature. After successive incubations (except the normal human serum block), the slides were washed twice with TBS for 5 minutes.
Assessment of CA IX Expression
Tumor Hormone Receptor Status
Assessment of Tumor Grade and Necrosis
Statistical Analysis
The clinicopathologic features of this unselected cohort of 103 women with invasive breast cancer are described in detail in Table 1. Among these patients, the median age was 59 years, the median tumor size was 2.4 cm, 58% had axillary node-positive disease, and 68% were positive for ER. Necrosis was present in 50 patients, and among this subset, marked necrosis (  25% of tumor) was present in 14 patients. The mean percentage necrosis was 17% (SD, ± 16%). The majority, 90 cases, were invasive ductal carcinomas; 11 were invasive lobular carcinomas, and two were tubular carcinomas. The median duration of follow-up for the entire cohort was 6.2 years; to date, 41 members (40%) of the cohort have relapsed and 32 (31%) have died.
CA IX Expression in Relation to Clinicopathologic Variables CA IX expression was detectable in 48% of cases as strong membranous staining within epithelial tumor cells. The pattern of expression was predominantly focal within the tumor and typically limited to tumor cells immediately adjacent to areas of necrosis. Expression was also occasionally seen adjacent to areas of dense collagenous stromal scar within the central regions of some tumors. The focal nature of expression was reflected in the distribution of IHC scores (range, 0 to 225; median, 0; mean, 23), with only 14% of tumors having an IHC score greater than 50. Representative examples of tumors showing low, moderate, and high CA IX expression are illustrated in Fig 1.
CA IX expression was compared with several established clinicopathologic prognostic variables (Table 2). Within the entire cohort, significant associations were found between the presence and level of CA IX expression and the presence of necrosis (P < .001), higher tumor grade (P = .02), and negative ER status (P < .001). There were no apparent relationships with other prognostic variables such as age, nodal status, tumor size, tumor type, and EGFR status. Necrosis was also assessed in relation to ER status and grade and was significantly associated with ER-negative status (P = .002) and high grade (P < .0001).
Relationship of CA IX to RFS and OS Univariate analysis of established prognostic factors and their relationship to survival confirmed that nodal status, grade, and ER status were all significantly related to both RFS (P values, < .001, = .04, and = .03, respectively) and OS (P values, < .001, = .04, and = .02, respectively). Tumor size was also predictive of RFS (P = .04), and patient age was found to be predictive of OS (P = .05). The presence of CA IX expression showed a significant association with a shorter RFS (P = .004) and a poorer OS (P = .001) (Fig 2). Similarly, the level of CA IX expression was also significant in terms of RFS (P = .04) and OS (P < .001). Interestingly, the presence of necrosis was also significantly associated with OS (P = .03).
In multivariate Cox proportional hazards analysis, where nodal status, ER status, tumor size, grade, and necrosis were considered together with the CA IX status, positive lymph node involvement was an independent predictor of RFS (hazard ratio, 2.90, P = .006). Although statistically insignificant, the presence of CA IX expression trended for a worse RFS (hazard ratio, 2.13, P = .06). When these same factors were analyzed for their impact on overall survival, only positive lymph node status and CA IX presence were significant independent predictors of OS (P = .004 and P = .05, respectively) (Table 3). The presence of any CA IX staining was found to be an independent poor prognostic factor for overall survival, with a hazard ratio of 2.61 (P = .05, 95% confidence interval, 1.01 to 6.75). Moreover, quantitatively, every 10-unit increase in CA IX IHC score was associated with a hazard ratio of 1.16 (P = .002, 95% confidence interval, 1.06 to 1.27). Thus, not only the presence but also the extent of CA IX expression is independently associated with a poorer outcome in this cohort of patients with breast cancer.
We have recently shown a marked upregulation of CA 9 mRNA and protein in cancer cell lines exposed to hypoxia.19 In this current study, we have extended our previous observation to show that CA IX is expressed in invasive breast carcinoma in vivo, with expression being virtually restricted to regions directly adjacent to areas of necrosis. Furthermore, we have demonstrated that CA IX is associated with several poor prognostic factors, including high tumor grade, ER-negative status, and the presence of necrosis. Finally, CA IX expression is predictive for reduced RFS and OS in univariate analysis and is an independent factor for overall survival in multivariate analysis. Though CA IX was not statistically an independent predictive factor for RFS in multivariate analysis, a trend for a worse RFS was seen (P = .06). Because this is what is to our knowledge the first published study describing a possible relationship to outcome for this carbonic anhydrase, further work in larger cohorts of patients is warranted to confirm these findings. CA IX was first identified in a human cervical carcinoma (HeLa) cell line and was subsequently found to be present in human carcinoma cell lines and clinical tumor specimens, but not in the corresponding normal tissue.22 Further evidence for a role in tumorigenesis came with the acquisition of many of the features of a transformed phenotype by stable transfection of NIH 3T3 cells with the MN cDNA (altered morphology, enhanced DNA synthesis with shorter doubling times, and capacity for anchorage-independent growth).23 Cloning of MN identified four distinct domains: an N-terminal region with structural features suggesting a role in extracellular matrix interactions, a second extracellular domain with close homology to the catalytic domain of other members of the alpha-CA gene family, a transmembrane region, and an intracytoplasmic C terminus.23,24 In the current study, CA IX expression was closely correlated with the presence of necrosis, believed to be an indicator of local hypoxia, within invasive breast tumors. In a recent study, conducted on a similarly sized cohort of invasive breast cancers, we found that the presence of necrosis correlated with high vascular density, high tumor grade, and ER-negative status.11 However, necrosis was not related to RFS or OS. In the present cohort, we have also confirmed that necrosis is associated with ER-negative and high-grade tumors, but necrosis also showed a significant relationship with poor overall survival, but not with RFS, in univariate analysis. The explanation for this difference is not clear; however, it is of potential relevance that the current cohort has longer follow-up (74 months v 63 months). Previous work on CA IX has identified a similar pattern of membranous expression on malignant cells, with rare expression in normal or benign tissue (most prevalent in the gastrointestinal tract and associated organs).28-32 In a study of 65 non–small-cell lung carcinoma specimens, the frequency of positive immunostaining was 80%, with no correlation with histologic subtype or tumor differentiation.31 In a series of 147 renal cell carcinomas, IHC demonstrated strong staining in 87% of tumors. Interestingly, there was a significant inverse relationship between MN/CA IX staining and tumor grade and stage, with a greater proportion of malignant cells staining positive in the lower-grade and -stage tumors.36 This inverse correlation of level of CA IX expression and histologic grade and other poor prognostic factors (eg, depth of invasion, lymph node metastasis) has also been shown in studies with cervical carcinoma,37 colorectal carcinoma,32 and esophageal cancer.30 This is different from the results of our study, where there is a positive correlation between the level of CA IX expression and the tumor grade, as indicated by both histologic grade and ER status. The reason for this difference is unknown. However, one possibility is that although hypoxia associated with visible necrosis seems to be a dominant factor in the regulation of CA IX in breast tissues, there may be other factors involved in the regulation CA IX expression in other tissues and related tumor types. Certainly an association between CA IX expression and areas of necrosis has not been described in these other studies. An explanation for the association seen in breast tumors between CA IX expression and poor prognosis may lie in the nature of its involvement in pH regulation in breast tissue. Evidence exists that the extracellular pH of solid tumors is often more acidic than that of normal tissue,38,39 whereas the intracellular pH in tumors is similar to normal cells.39-41 It is thought that this altered cellular pH gradient may have implications for resistance to certain chemotherapeutic agents, in particular weakly basic drugs such as doxorubicin, because of reduced permeability and cell uptake. In fact, a recent study has demonstrated enhanced efficacy for doxorubicin in human breast cancer cells in vitro and in vivo when the pH is raised from 6.8 to 7.4.42 Because carbonic anhydrases exert their enzymatic effect by catalyzing the reversible interconversion of carbonic acid to carbon dioxide, expression of CA IX may reflect an adaptive cellular response to extremes of pH and therefore may indicate those tumors with regions of very low extracellular pH that may then contribute to chemotherapeutic and radiation resistance. Alternatively, it has been proposed that expression of CA IX may positively contribute to reduction of extracellular pH and thereby contribute as a causal factor to increased malignancy and invasiveness.43 Last, the utility of CA IX as a biomarker may have both diagnostic and therapeutic implications. Besides its specificity by IHC for malignant cells, a recent study has shown that reverse transcriptase–polymerase chain reaction detection of CA 9 can be highly specific for detecting circulating renal carcinoma cells.44 Likewise, work has already been performed on a radiolabeled antibody to CA IX (mAbG250) for scintigraphic diagnosis of renal cell carcinoma,45 and this same antibody has also been shown to exert antitumor effects on a renal cell carcinoma xenograft model.46 However, the specific functions of CA IX on the growth of breast cancer remain to be established. In conclusion, we have shown that CA IX expression is closely associated with necrosis in invasive breast carcinoma, complementing previous work demonstrating that CA IX is regulated by hypoxia in vitro. We have also shown that this biomarker has prognostic significance for a shorter RFS and a worse OS in an unselected series of invasive breast carcinomas. It remains to be determined whether the expression of CA IX in breast cancers in vivo is simply an indicator of response to necrosis caused by local hypoxia or whether it actively contributes to the process of tumor progression. Confirmation of the prognostic significance of CA IX expression in larger randomized studies is clearly warranted to explore this relationship further.
S.K.C. is supported by the Shane Fellowship and the Canadian Breast Cancer Foundation, British Columbia/Yukon Chapter. P.H.W. is the recipient of a Medical Research Council of Canada Scientist Award, an Academic Award from the United States Army Research and Material Command, and travel awards from Burroughs Welcome Trust and the Royal College of Physicians and Surgeons of Canada.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
