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ABSTRACT: Angiogenin (ANG) is a potent angiogenic factor that is up-regulated by hypoxia. ANG expression is well documented in normal tissues and in common tumours, but its expression has not been reported in the normal human kidney or in Wilms' tumours (WT). We examined ANG expression in WTs, human fetal kidney (FK) and childhood kidney (NK) samples and studied its relationship with microvascular density (MVD) and with three other hypoxia-induced angiogenic factors: lactate dehydrogenase A (LDHA), vascular endothelial growth factor (VEGFA) and BHLHE40 (basic helix-loop-helix transcription factor E40). Total ANG protein levels were significantly lower in WTs when compared with those in 15 matched-paired NKs. ANG immunoreactivity was observed in the glomeruli, proximal tubules and vessels in the FKs and NKs, indicating that ANG plays a physiological role in the human kidney. ANG cellular localization and distribution in 27 WTs reflected the pattern observed in the FKs. ANG colocalized with LDHA in the perinecrotic areas of untreated WTs suggesting up-regulation by hypoxia. There was a significant correlation between CD31-MVD and ANG-MVD. ANG, CD31, VEGFA and BHLHE40 mRNA levels were significantly lower in 15 WTs compared with matched-paired NKs. Univariable and multivariable statistical analyses showed significant correlations between ANG and CD31, ANG and BHLHE40 mRNAs and a weaker relationship between ANG and VEGFA mRNAs. ANG expression in WTs recapitulates that seen during nephrogenesis, and correlation with CD31-MVDs and mRNAs is consistent with a contribution to angiogenesis in WTs. Our study contributes to the understanding of angiogenesis during development and in WTs.
International Journal of Experimental Pathology 02/2013; · 2.57 Impact Factor
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ABSTRACT: To study the expression of hypoxia-inducible factor 1α (HIF-1α) in clinical neuroblastoma (NB) samples and its association with the clinicopathological features, biological features and survival of NB patients.
Immunohistochemistry indicated that elevated HIF-1α expression was present in 30 of 90 (33%) NBs. This expression was correlated significantly and positively with higher clinical stage (P = 0.002), ≥18 months of age at presentation (P = 0.020), high-risk group (P = 0.005), unfavourable pathology (P = 0.002), MYCN amplification (P < 0.001), 1p deletion (P = 0.004) and 17q gain (P = 0.002). Enzyme-linked immunosorbent assays showed that total HIF-1α protein was significantly higher in NBs of patients with all examined adverse prognostic factors except for age. Univariate survival analysis revealed that higher-than-median HIF-1α total protein levels were associated significantly with a decrease in event-free survival (EFS) (P = 0.017), but not in overall survival (OS) (P = 0.12). HIF-1α immunoexpression by ≥10% of tumour cells was associated significantly with decreased OS and EFS (P = 0.002 and P = 0.004, respectively), but not in multivariate analysis after adjusting for the high-risk group (P = 0.16 and P = 0.19, respectively).
HIF-1α was increased significantly in patients with NB associated with unfavourable characteristics. HIF-1α is a prognostic indicator of poor OS and EFS and defines subgroups of NBs with aggressive clinical behaviour.
Histopathology 05/2012; 61(3):417-27. · 3.08 Impact Factor
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ABSTRACT: The overexpression of carbonic anhydrase IX, a hypoxia-induced enzyme, is associated with an adverse prognosis in many cancers. However, carbonic anhydrase IX expression in neuroblastoma, the most common extracranial pediatric tumor, has not been reported. Membranous and/or strong cytoplasmic carbonic anhydrase IX expression, assessed by immunohistochemistry, was present in 21 (23%) of 91 neuroblastomas but was absent in ganglioneuromas (n = 9). The proportion of neuroblastomas showing membranous carbonic anhydrase IX expression was higher in neuroblastomas with 1p deletion and MYCN amplification. Nuclear carbonic anhydrase IX expression was seen in less than 10% of ganglion cells in all ganglioneuromas. Of 91 neuroblastomas, 16 (18%) showed nuclear carbonic anhydrase IX expression in 10% or more tumoral cells. The proportion of neuroblastomas showing nuclear carbonic anhydrase IX expression was significantly higher in patients with adverse clinical (increasing stage and high-risk group), pathologic (unfavorable group and high mitosis-karyorrhexis-index), and biologic (MYCN-amplification and 1p deletion) factors. Carbonic anhydrase IX total protein levels, quantified by enzyme-linked immunosorbent assay, were higher in neuroblastomas (n = 49; geometric mean, 0.47 pg/µg; range, 0.0-6.52 pg/µg) than in ganglioneuromas (n = 6; geometric mean, 0.20 pg/µg; range, 0.09-0.47 pg/µg) and were significantly higher in MYCN-amplified neuroblastomas. Nuclear carbonic anhydrase IX expression was associated with a poorer overall survival (P = .003) and event-free survival (P = .004), although the relationships were no longer significant when adjusted for high-risk disease. There was no significant association of membranous carbonic anhydrase IX expression or higher-than-median total protein levels with overall survival or event-free survival. Carbonic anhydrase IX is expressed at significantly higher levels in neuroblastomas from patients with adverse clinicopathologic and biologic factors indicating that it is a biomarker of aggressive disease in neuroblastomas.
Human pathology 03/2012; 43(10):1651-60. · 3.03 Impact Factor
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ABSTRACT: 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.
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).
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.
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.
BMC Cancer 09/2011; 11:390. · 3.01 Impact Factor
