Overexpression of carbonic anhydrase and HIF-1α in Wilms tumours

BACKGROUND: Overexpression of carbonic anhydrase (CA IX) is associated with poor survival in several adult-type cancers but its expression is undocumented in Wilms tumour (WT), the most common tumour of the paediatric kidney. METHODS: CA9 expression was measured using polymerase chain reaction (PCR) in 13 WTs and matched-paired non-neoplastic kidneys (NKs). CA IX and hypoxia-inducible factor-1 α-subunit (HIF-1α) protein were quantified in 15 matched-paired WTs and NKs using enzyme-linked immunosorbent assays. CA IX and HIF-1α were localised by immunostaining tissue sections of 70 WTs (untreated WTs, n = 22; chemotherapy-treated WTs, n = 40; relapsed/metastatic WTs, n = 8). CA IX-positive untreated WTs (n = 14) were immunostained for vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT1) and CD31. Double staining for CA IX and CD31 was performed in WTs (n = 14). RESULTS: CA9 full length (FL) was significantly up-regulated in WTs compared to NKs (p = 0.009) by real-time PCR. Conventional PCR showed expression of alternative splice variant in all NKs and WTs but FL in WTs only. WTs showed a 2-fold increase in CA IX protein over NKs (p = 0.01). HIF-1α levels were up-regulated in WTs compared to NKs, although the difference was not statistically significant (p = 0.09). CA IX and HIF-1α immunolocalisation were observed in 63% and 93% of WTs, respectively. The median fraction of cells staining positively for CA IX and HIF-1α was 5% and 22%, respectively. There was no significant association between the expression of either CA IX or HIF-1α and clinicopathological variables in WTs resected following chemotherapy. VEGF and GLUT1 immunoreactivity was seen in 94% and 100% with the median fraction of 10% and 60% respectively. Co-expression and co-localisation of all four hypoxia markers was seen in 7/14 and 6/14 cases respectively. CA IX was seen in well vascularised areas as well as in the peri-necrotic areas. CONCLUSIONS: Carbonic anhydrase 9 (mRNA and protein), and HIF-1α protein are overexpressed in a significant portion of WTs. No significant association was detected between the expression of either CA IX or HIF-1α and clinicopathological variables in WTs resected following chemotherapy. Cellular localisation studies in untreated WTs suggest that CA IX and HIF-1α are regulated by hypoxia and non-hypoxia mechanisms.