High levels of polo-like kinase 1 and phosphorylated translationally controlled tumor protein indicate poor prognosis in neuroblastomas.

Despite multimodality treatment, the long-term survival of high-risk patients with neuroblastomas is below 50%. New anti-mitotic drugs against targets, such as polo-like kinase 1 (PLK1), are being evaluated in early phase clinical trials. PLK1 phosphorylates the translationally controlled tumor protein (TCTP). We investigated the expression of PLK1 and the phosphorylated substrate, pTCTP, by immunostaining eighty-eight neuroblastomas. Digitally scanned slides were scored using image analysis software. The median PLK1 and pTCTP proliferation indices (PIs) were 4.6 and 1% respectively. There was moderate positive correlation between PLK1 and pTCTP (ρ = 0.65). The PIs for both markers were significantly higher in neuroblastomas from patients with adverse clinical (advanced-stage, high-risk group, primary abdominal compared to extra-abdominal sites), biological (MYCN amplification, 1p deletion, 17q gain) and pathological (undifferentiated or poorly differentiated status, high mitosis-karyorrhexis index, [MKI], unfavorable histology) factors. Using Cox regression models, higher-than-median PLK1 and pTCTP PIs were associated with a shorter overall survival (OS) and event-free survival (EFS) in the univariate analyses. In the multivariate analyses, a high PLK1 PI count was associated with significantly shorter OS and EFS, independent of MYCN amplification and MKI; in addition, the significantly shorter EFS was independent of the risk-group. After adjustment for MKI and MYCN amplification, and for risk-group, high pTCTP PI was also associated with significantly shorter OS. Our study shows that PLK1 provides valuable prognostic information in patients with neuroblastomas.

High proliferation index, as determined by immunohistochemical expression of Aurora kinase B and geminin, indicates poor prognosis in neuroblastomas.

Expression profile analysis of cell cycle biomarkers provides a powerful index of the proliferative state of tumors, which is linked to disease aggressiveness. We investigated the impact of the biomarkers of S-G2-M phases of cell cycle, Aurora kinase B (AURKB) and geminin (GMNN), on disease progression in neuroblastomas. The expression of AURKB and GMNN was studied by immunostaining 84 neuroblastomas. A proliferation index (PI) was obtained on scanned immunostained slides using image analysis software. The median PI was 8.5 % for AURKB- and 16.8 % for GMNN-stained slides with a high correlation between the two (r s = 0.72, P < 0.001). The PI for both markers was significantly higher in neuroblastomas from patients with unfavorable clinical (high-risk group, advanced stage, age ≥18 months at presentation, primary abdominal compared to extra-abdominal sites), biological (MYCN amplification, 1p deletion, 17q gain), and pathological (undifferentiated or poorly differentiated status, high mitosis-karyorrhexis index, [MKI], unfavorable histology) factors. Using Cox regression models, a higher-than-median AURKB and GMNN PI was associated with a significantly shorter overall survival (OS) and event-free survival (EFS) in univariable analysis. In multivariable analysis, a high AURKB PI was associated with significantly shorter OS and EFS, independent of MYCN amplification, and significantly shorter EFS, independent of MKI. High GMNN PI was also associated with significantly shorter OS and EFS after adjusting for MYCN amplification but failed to reach statistical significance after adjusting for MKI. Our study shows that in neuroblastomas, AURKB- or GMNN-based PI provides valuable prognostic information and high PI indicates aggressive disease.

High phosphohistone H3 expression correlates with adverse clinical, biological, and pathological factors in neuroblastomas.

Phosphohistone H3 (pHH3), a biomarker of the late G2- and M-phase of the cell cycle, provides a powerful indication of the proliferative state of many cancers. We investigated the prognostic significance of pHH3 by immunostaining 80 neuroblastomas and counting the average number of strongly stained nuclei and mitotic figures. The median and 75th percentile pHH3 proliferation indices (PIs) were 0.54% and 1.06% (range, 0.01% to 2.23%) respectively. pHH3 expression was significantly higher in neuroblastomas from patients with adverse clinical characteristics, all unfavorable pathological factors including high mitosis karyorrhexis index (MKI), and adverse biological factors including MYCN oncogene amplification. High pHH3-PIs, at 1% threshold, were significantly associated with a shorter overall survival (OS) and event-free survival (EFS) in the univariable Cox regression analyses. In the multivariable models, high pHH3 counts were significantly associated with worse OS after adjustment for age but were not independent of either high MKI or MYCN amplification. In children less than 18 months of age, high MKIs and high PHH3-PIs were associated with significantly worse OS and EFS. In conclusion, high pHH3 expression correlates strongly with high MKI and MYCN amplification and indicates poor prognosis in neuroblastomas.

Angiogenin expression in human kidneys and Wilms' tumours: relationship with hypoxia and angiogenic factors.

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.