Distribution of pediatric brain tumor cases
As shown in Table , the most common brain tumor in the patient cohort was the pilocytic astrocytoma, accounting for 36% of the tumors studied. However, infiltrating astrocytomas (i.e., all astrocytoma WHO II, III, and IV) as a group exceeded the number of pilocytic astrocytoma cases, accounting for 42% of the cases studied. Astrocytomas are therefore slightly over represented in this cohort compared to that reported in the CBTRUS, in which pilocytic astrocytomas (WHO grade I) comprise 14–20% of tumors and infiltrating astrocytomas range from 11–15% of tumors [2
Anatomical site of the tumors is listed in Table , and the distributions of tumor type per anatomical site is illustrated in Fig. . The most common tumor site was the posterior fossa (26.7%), followed by the cerebral hemispheres (in toto, 25.6%). As the specific proportion of cortical based and cerebellar based brain tumors are slightly increased relative to the CBTRUS population (18 and 16%, respectively), these tumor sites are overrepresented in this cohort. Infiltrating astrocytomas (WHO Grades II–IV) accounted for the majority of the tumors arising in the cerebral hemispheres, while the majority of posterior fossa tumors were pilocytic astrocytomas (Fig. ).
Distribution of OLIG2 expression in pediatric tumors
All astrocytic tumors, including all pilocytic astrocytomas (WHO Grade I) and the diffuse-type astrocytomas (WHO Grades II–IV) showed diffuse OLIG2 expression (Table ). The glioblastomas and the pilocytic astrocytomas had the highest mean OLIG2 score, followed by the diffuse-type infiltrating astrocytomas WHO grade II–III. The primary ependymomas (including anaplastic ependymoma, WHO grade III) and two subependymal giant cell astrocytoma were noteworthy for the absence of OLIG2+ cells. Evaluation of adult ependymomas also showed a near absence of OLIG2+ tumor cells (i.e., non-recurrent adult ependymoma WHO II and non-recurrent pediatric ependymoma WHO II were OLIG2 score = 0). The recurrent anaplastic astrocytomas (WHO grade III) and the recurrent cellular ependymomas tended to have a higher number of OLIG2+ cells in comparison to the corresponding primary tumors whereas the recurrent lower grade astrocytic tumors (WHO grade I and II) had much lower numbers of OLIG2+ cells. Analysis of variance (ANOVA) was performed to test the significant differences between OLIG2 expressions amongst the tumor types. The P-values of statistically significant tumor comparisons derived from intergroup comparison (performed by ANOVA/Tukey HSD test) are listed in Table . In summary, OLIG2 expression in SEGA and ependymomas were significantly lower than the astrocytic neoplasms.
Figure shows representative fields of the astrocytic tumors: pilocytic astrocytoma, infiltrating astrocytoma WHO grade II–III. Many of the pilocytic astrocytoma cases showed near universal, diffuse OLIG2 expression, as illustrated in Fig. b, left panel. Glioblastoma, as a group showed intense OLIG2 expression, but the distribution patterns of the immunoreactive cells varied. The most common pattern was a relatively diffuse expression in single cells or small cell clusters as shown in Fig. b. One glioblastoma arising in the thalamus, illustrated in Fig. d, demonstrated a biphasic pattern of OLIG2 expression with majority of the OLIG2 immunoreactive nuclei in the more poorly differentiated cells that were distributed in highly cellular zones while the more differentiated astrocytic phenotypes were OLIG2 negative. These poorly differentiated, OLIG2+ cells were also immunoreactive for GFAP (Fig. d, inset). When an infiltrating edge of the glioblastomas was available for analysis, OLIG2 positive tumor cells were present in the populations of tumor cells infiltrating the surrounding brain; however, in contrast to the overall tendency for localization of the tumor cells in perineuronal and perivascular zones, OLIG2+ cells did not show a preferential perineuronal satelitosis or perivascular structuring (Figs. e, f).
A total of 16 pediatric ependymomas were tested for OLIG2 expression. Representative photographs of OLIG2 stained ependymomas are shown in Fig. . The majority of cases (13 of 16) showed no significant OLIG2 expression (score = 0). One case of myxopapillary ependymoma showed a diffuse, strong expression of OLIG2. A second case of myxopapillary ependymoma showed no OLIG2 expression. Discrete zones of OLIG2+ cells were present in one case of recurrent cellular ependymoma in an 8-year old boy (see Fig. d) and in one case of a 17-year old boy with a fourth ventricular anaplastic ependymoma, WHO III (data not shown). The OLIG2 staining pattern in the recurrent pediatric ependymoma is more typical of that described in adult ependymomas [23
]. Taken together, 18.8% of pediatric ependymoma cases showed at least some OLIG2 expression, a result similar to that reported by other investigators [35
]. Comparison of OLIG2 expression in adult ependymomas showed similar results to the pediatric ependymomas (mean OLIG2 score = 0). No adult myxopapillary ependymomas (0 of 3 total cases) showed significant OLIG2 expression.
Tumor type and anatomic site affecting OLIG2 expression
Although the level of OLIG2 expression appeared to vary according to tumor site (Table ), this trend usually resulted from the disparity of specific tumor types that were associated with particular anatomic zones. However, the anaplastic astrocytomas that arose in the occipital lobes had low OLIG2 expression compared to other sites, and regardless of tumor type, tumors arising in the deep supratentorial midline structures (suprasellar, optic nerve, thalamus) typically had high levels of OLIG2 expression. The anatomical site that showed the highest OLIG2 expression was the deep cerebral gray matter (average OLIG2 score of 2.7). The anatomical sites with the lowest OLIG2 expression were intraventricular tumors (average OLIG2 score = 0.66). Intraventricular tumors were chiefly composed of low OLIG2 expressing tumors such as SEGA and ependymoma. Tumors arising in the frontal/temporal/parietal cerebral cortex show a large variation in OLIG2 expression (standard deviation of OLIG2 score = 1.2). Most astrocytic neoplasms arising in frontal/temporal/parietal cerebral cortex showed diffuse levels of OLIG2 expression; however, three cases of anaplastic ependymoma, WHO III showed low OLIG2 expression (OLIG2 score = 0), which accounts for the large variability (standard deviation = 1.2). Statistical analysis by ANOVA and Tukey HSD test demonstrates that only ventricle-brainstem, ventricle-deep deep gray matter, and ventricle-posterior fossa comparisons were statistically significant (P = 0.005, 0.0009, 0.002, respectively). OLIG2 expression differences between all other sites were not statistically significant (in all instances, P > 0.05).
Cellular proliferation and OLIG2+ expression
OLIG2 regulates replication competence in a genetically relevant murine model [9
]. To test if proliferating cells were OLIG2 positive, the proportion of Ki67+ cells that were also OLIG2+ were determined in selected cases of pilocytic astrocytoma, infiltrating astrocytoma WHO grade II, anaplastic astrocytoma WHO grade III, glioblastoma WHO grade IV, and ependymoma WHO grade II. Dual labeling immunohistochemistry for OLIG2 and Ki-67 was performed without a hematoxylin counterstain. Hence, to obtain a surrogate Ki67 labeling index in the cases examined, the proportion of OLIG2+ cells that are also Ki67+ was determined. Results for astrocytoma are listed in Table , and examples of dual labeling are shown in Fig. . Glioblastoma had the highest percentage of OLIG2 expressing cells that were also Ki-67 positive (i.e., mean (number of OLIG2+ Ki67+ cells)/total Ki67+ cells = 16.3%) and also the highest subpopulation of OLIG2− cells within the fraction of proliferating cells (i.e., mean (number of OLIG2-Ki67+ cells)/total Ki67+ cells = 15.5%). Most of the proliferating (Ki-67 positive) cells in the diffuse-type astrocytomas (WHO grade II-III) were also OLIG2+ (i.e., (number of OLIG2+ Ki67+ cells)/total Ki67+ cells = 92–94%). However, the mean proportion of OLIG2+ cells that were also Ki67+ (i.e., (number of OLIG2+ Ki67+ cells)/total OLIG2+ cells) varied from 1.0–16.3%. The mean proportion of OLIG2+ cells that were also Ki67+ showed a trend to being associated with increasing tumor grade. As expected, statistical analysis by ANOVA/TukeyHSD test showed the mean proportion of OLIG2+ cells that were also Ki67+ in glioblastoma multiforme WHO IV to be significantly different from pilocytic astrocytoma, astrocytoma WHO II, and anaplastic astrocytoma WHO III (P
= 0.007, 0.0008, 0.0004, respectively). This finding is in concurrence with data showing a higher Ki67 labeling index in glioblastoma relative to lower grade astrocytomas.
OLIG2 and Ki67 dual quantifications in pediatric human gliomas
Even though the overall rate of cell proliferation in pilocytic astrocytomas was very low, about 85% of the proliferating cells were also OLIG2+ , which represented only about 1.6 percent of all cells expressing OLIG2. All the ependymoma cases that were OLIG2− showed scattered Ki67 positive nuclei throughout the tissue sections (data not shown). The average percentage of Ki67 + cells that were also OLIG2 + were 84.6% (st. err. = 6.2) for pilocytic astrocytoma, 94.3% (st. err. = 5.7) for grade II astrocytoma, and 92.3% (st. err. = 2.4) for anaplastic astrocytoma WHO grade III. Statistical analysis by ANOVA did not demonstrate any significant difference between astrocytoma groups but did show difference between astrocytoma-ependymoma groups.
BRAF mutation shows no correlation with OLIG2 expressing tumors
Evaluation of BRAF mutation status in a subset of pediatric gliomas is presented in Table . To test an association between OLIG2 score and BRAF mutation, contingency tables were created and analyzed by the Fisher Exact Test. Groups were separated into BRAF mutated (which included BRAFV600E missense mutations and KIAA1549-BRAF fusion transcripts) and BRAF non-mutated. The OLIG2 contingency table separated the patients listed in Table into patients with an OLIG2 score of 3 (BRAF mutated n = 6, BRAF non-mutated n = 4), and an OLIG2 score <3 (BRAF mutated n = 0, BRAF non-mutated n = 2). No statistically significant association between OLIG2 expression and BRAF mutation was determined (P = 0.45). Tumor type contingency tables were constructed for pilocytic astrocytoma (BRAF mutated n = 4, BRAF non-mutated n = 0), astrocytoma WHO II ((BRAF mutated n = 1, BRAF non-mutated n = 3), anaplastic astrocytoma WHO III (BRAF mutated n = 1, BRAF non-mutated n = 2), and glioblastoma (BRAF mutated n = 0, BRAF non-mutated n = 1). No statistically significant correlation was identified between the presence of a BRAF mutation and tumor diagnosis (P = 0.11). However, a statistically significant association between the presence of KIAA1549-BRAF fusion transcripts in pilocytic astrocytoma and its absence in the other tumor groups was noted (P = 0.006).
OLIG2 expression and BRAF analysis in pediatric gliomas
In silico analysis of ependymoma and astrocytoma tissue microarray datasets
Pilocytic astrocytoma and ependymoma transcriptional expression datasets obtained from previously published Affymetrix expression microarrays were evaluated (see methods). Quality control assessment of the ependymoma and pilocytic astrocytoma datasets demonstrated similar average probe signal intensities and high intergroup correlation (Supplementary Fig. 1). In each group, one of three arrays showed high RNA degradation. Significant differential expression was noted in 1402 of 53272 genes (significance threshold was set to P < 0.05 by t-test). The genes that were most significantly differentially expressed in the two datasets are listed in Table . The P value listed is an adjusted P value derived after Benferroni correction of the limFit/TopTable P-value. Of interest, SH2 showed significant differential expression with _s_at probes. The _s_at is predicted to bind to more than one transcript of the same gene family, suggesting that gene family members are differentially expressed in these two tumors.
Transcriptional comparison of OLIG2
genetically null versus wild-type neural stem cells in mice demonstrated differential expression in multiple E-box containing genes [9
]. This list of the differentially expressed genes reported by Ligon et al. [9
] was used to test for differential expression between the pilocytic astrocytoma and ependymoma microarray data. No statistically significant differential expression of these E-box containing genes was noted when comparing pilocytic astrocytoma and ependymoma array data (in all instances, P
> 0.05 by t
Table lists neural cell lineage associated genes for neural stem cells, astrocytes, oligodendrocytes, and ependymal cells. SOX2
and Aquaporin 4
showed significant increased expression in ependymoma relative to pilocytic astrocytoma. Of note, the _s_at probe for Aquaporin 4
showed no statistically significant difference between the ependymoma and pilocytic astrocytoma datasets (P
= 0.17), whereas the _at showed significant difference (P
= 0.005); this suggests that pilocytic astrocytomas may express other genes of a similar family, but do not express the Aquaporin 4 transcript. Relative to ependymomas, pilocytic astrocytomas showed significantly increased expression of nestin
, and Oligodendrocyte Myelin Glycoprotein
). Of note, PLP1
showed elevated RMA expression measures in pilocytic astrocytoma relative to ependymoma, but the P
value was slightly above the threshold for significance set for this study (P
= 0.06). Increased expression of Rootletin
, a structural protein present in the cilia of ependymal cells [38
], was significantly higher in ependymoma relative to pilocytic astrocytoma. In contrast to pilocytic astrocytoma, evaluation of the pediatric glioblastoma expression dataset [11
] showed minimal expression of OMG
(mean expression measure = 416.2(81.3)).1
, and GFAP
were highly expressed in pediatric glioblastoma (mean expression measures 1085.9 (117.1), 2609.4 (426.5), 5499.8 (731), respectively) (see footnote 1).