Table 1.
Demographic data from patients analyzed in this study.
Figure 1.
Expression levels of genes in diffusely infiltrative astrocytomas (AGII to GBM).
Transcript levels of ID4 (A), SOX2 (B), SOX4 (C), OCT-4 (D), NANOG (E) and CD133 (F) were determined in 26 low-grade astrocytomas (AGII), 18 anaplastic astrocytomas (AGIII) and 86 GBM cases relative to 22 non-neoplastic (NN) by quantitative real-time PCR. Relative expression values were calculated based on the geometric mean of HPRT, GUSB and TBP expression levels of each sample and non-neoplastic brain values. The following equations were applied to calculate gene relative expression according to primer efficiency (E) in tumor samples versus the mean of non-neoplastic tissues: 2−ΔΔCt [27] for SOX2, SOX4, OCT-4 and CD133; and 1+E−ΔΔCt [28] for ID4 and NANOG, where ΔCt = Ct specific gene – mean Ct of housekeeping genes and ΔΔCt = ΔCt tumor – mean ΔCt non-neoplastic. Red dots represent the secondary GBM cases. Horizontal bars show the median of each group and the values are presented in Table 2. NANOG expression in 15 NN and 40 GBM cases was very low and, as a result, the horizontal bar for NN does not appear in the graphic (median = 0). The difference of relative gene expressions among the groups were statistically significant (p<0.0005 for ID4, SOX2, SOX4, OCT-4 and NANOG; and p<0.05 for CD133, Kruskal-Wallis test). A pair-based comparison was assessed using Dunn test. The p value results are shown, where ***p<0.0005, **p<0.005 and *p<0.05.
Figure 2.
Correlation between ID4 and SOX2, SOX4, OCT-4, NANOG and CD133 expression levels in diffusely infiltrative astrocytomas.
Correlation was assessed in AGII (A, D, G, J, M), AGIII (B, E, H, K, N) and GBM (C, F, I, L, O) cases. ID4 expression level was correlated to SOX2 (A-C), SOX4 (D-F), OCT-4 (G-I), NANOG (J-L) and CD133 (M-O) expression levels. The significant correlations are shown in black and the non-significant in grey. r correlation coefficient assessed by Spearman-rho test, and r* by Pearson’s correlation test.
Table 2.
Median of relative expression levels of the analyzed genes in astrocytomas, according to morphology.
Figure 3.
Comparison of gene expression levels between the wild-type TP53 (WT TP53) and the mutated TP53 (Mutated TP53) in AGII cases.
Higher expressions of ID4 (A), SOX2 (B), SOX4 (C) and NANOG (E) were observed on the mutated TP53 AGII cases. No difference was found for OCT-4 (D) and CD133 (F) relative expression between the two groups. White lozenges represent the deceased patients. The p values are: *p<0.05 and ** p<0.005, Mann-Whitney test.
Figure 4.
ID4, SOX2 and SOX4 immunohistochemistry.
Representative cases of wild-type TP53 AGII (A-C), mutated TP53 AGII (D-F), primary GBM (G-I) and secondary GBM (J-L) stained for ID4, SOX2 and SOX4 are demonstrated. Both mutated AGII and secondary GBM cases showed stronger and larger number of nuclear stained cells (score 3 for intensity and ≥75% of positive cells) for ID4, SOX2 and SOX4. Comparatively, wild-type TP53 AGII and primary GBM presented score 1 for intensity and <25% of positive cells. The reaction was performed in paraffin embeded tissue sections with a commercial polymer kit (Novolink; Novocastra, UK), using diaminobenzidine as developer and Harris hematoxylin for nuclear counterstaining. 200× magnification for all images.
Figure 5.
Heatmap displaying the relative gene expressions in low-grade astrocytoma (AGII), anaplastic astrocytoma (AGIII) and GBM cases according to TP53 mutation status.
The TP53 mutated cases are represented by side dashes. The mutated TP53 AGII cases showed more elevated expression levels of ID4, SOX2, SOX4 and NANOG. CD133 expressions were more heterogeneous among the cases. SOX2 and SOX4 showed similar expression levels to ID4. Similarly, secondary GBM cases also presented higher ID4, SOX2, SOX4 expression levels. OCT-4, NANOG and CD133 expression levels were heterogeneous among secondary GBM cases, and OCT-4 presented higher mRNA levels in primary GBM.
Figure 6.
Survival curve of GBM patients.
Twenty-five GBM cases out of 86 GBM cases presented concomitant high or low ID4, SOX4 and OCT-4 relative expresssion levels (12 GBM cases presenting high expressions and 13 low expressions for the three genes). The survival time difference between the two groups was statistically significant (log rank p-value = 0.014), presenting median survival time of 6 months for GBM cases presenting concomitant high expressions for the three genes compared to 18 months for GBM cases with low expressions.