Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenicpathways and independently to poor outcome in non-small cell lung cancer

Carbonic anhydrase-9 (CA9), a transmembrane enzyme with an extracellular ac tive site, is involved in the reversible metabolism of the carbon dioxide t o carbonic acid. Up-regulation of CA by hypoxia and the hypoxia-inducible f actor (HIF) pathway has been recently postulated (Wykoff et al. Cancer Res. , 60: 7075-7083, 2000). In the present study we examined the expression of this enzyme in non-small cell lung cancer. Of 107 cases analyzed, 39 (36.4% ) had strong membrane/cytoplasmic expression of CA9 and were grouped as pos itive. The staining was confined around areas of necrosis, and a significan t association of CA9 expression with the extent of necrosis was noted (P = 0.004). Nevertheless, 38 of 74 cases with focal or extensive necrosis did n ot express CA9. CA9 expression was more frequent in the squamous cell histo logy (P = 0.001) and with advanced T stage (P = 0.009). A significant coexp ression of CA9 with platelet-derived endothelial cell growth factor and bas ic fibroblast growth factor receptor expression was noted. Double staining of CA9 with anti-CD31 monoclonal antibody revealed an overall higher microv essel density in the areas expressing CA9 than in negative areas (P = 0.000 5). Thirty-one of 38 CA9-positive cases were positive for HIF1a/HIF2a, but HIF positivity was a more common event (68 of 107) and their patterns of ex pression were diffuse (not confined in the necrotic areas). A direct associ ation of CA9 expression with epidermal growth factor receptor, c-erbB-2, an d MUC1 expression was also noted (P < 0.04). Survival analysis showed that CA9 expression is related to poor prognosis. CA9 expression in tumors with low vascularization defined a prognosis similar to the one of patients with highly angiogenic tumors. Multivariate analysis revealed that CA9 expressi on is a significant prognostic factor independent of angiogenesis. We concl ude that CA9 is an important molecule in non-small cell lung cancer, the up -regulation of which occurs in highly hypoxic/necrotic regions of the tumor s. The expression of CA9 is linked to the expression of a constellation of proteins involved in angiogenesis, apoptosis inhibition, and cell-cell adhe sion disruption, which explains the strong association of CA9 with poor out come.