Methods for the detection of breast cancer have improved markedly over the past decade and patients are subsequently presenting with ever earlier stages of disease (Ernster and Barclay, 1997). Although established markers of tumour behaviour, including tumour size and nodal status are applicable for this group of patients, tissue-based markers that can predict the risk of progression and recurrence would improve the ability to identify those who would benefit from the treatment (Bennington et al, 1992; Ernster et al, 1996; Tabar et al, 2000). Indeed, several such markers, such as tumour grade and estrogen receptor (ER) status are well established. These factors mostly relate to cellular differentiation, while alternative indicators of the metabolic status of tumour cells, such as oxygenation, are relatively sparse (Brizel et al, 1997; Vaupel and Hoeckel, 1999). Necrosis is one such indicator of poor oxygenation and has been shown to be a prognostic indicator (Leek et al, 1999). However, necrosis is nonspecific, and may not always be associated with severe hypoxia (Mueller-Klieser et al, 1983; Parliament et al, 1997; Ramanujan et al, 2000). To identify endogenous hypoxic markers that might have utility as prognostic indicators, we and others have begun to examine the expression patterns of known hypoxia-inducible genes in tumour specimens. Potential markers include the oxygen-regulated subunits of the transcriptional complex hypoxia-inducible factor-1 (HIF-1), a key mediator of the hypoxic response, HIF-1α and HIF-2α, or HIF-1 target genes (Shweiki et al, 1992; Maxwell et al, 1997; Zhong et al, 1999).
Recently, we extended the range of HIF-1 target genes by identifying the two tumour-associated transmembrane carbonic anhydrases (CA) CA9 (Opavsky et al, 1996; Ivanov et al, 1998) and CA12 (Ivanov et al, 1998; Tureci et al, 1998) as upregulated by hypoxia in a range of epithelial cancer cell lines (Wykoff et al, 2000). Carbonic anhydrases catalyse the reversible hydration of carbon dioxide to carbonic acid, providing a potential link between metabolism and pH regulation. We were therefore interested in examining the expression of these CA in tumourigenesis and progression in breast cancer. Our initial studies focused on CA IX as this gene demonstrated a more dramatic hypoxic induction in the tumour cell lines examined (Wykoff et al, 2000). CA IX expression was closely correlated with the presence of necrosis in both in situ and invasive cancer (Chia et al, 2001; Wykoff et al, 2001), and was found to be an independent predictor of relapse-free and overall survival (Chia et al, 2001). In the present study, we have investigated CA XII expression in breast cancer in the anticipation that its expression might also serve as an indicator of tissue hypoxia and tumour progression. Specifically, we wished to assess the pattern of expression in invasive breast carcinoma and the relation to outcome.