Expression of oligodendroglial and astrocytic lineage markers in diffuse gliomas: use of YKL-40, ApoE, ASCL1, and NKX2-2

The phenotypic heterogeneity of astrocytic and oligodendroglial tumor cells complicates establishing accurate diagnostic criteria, and lineage-specific markers would facilitate diagnosis of glioma subtypes. Based on data from the literature and from expression microarrays, we selected molecules relevant to gliogenesis and glial lineage specificity and then used immunohistochemistry to assess expression of these molecules in 55 diffuse gliomas, including 8 biphasic oligoastrocytomas, 21 oligodendrogliomas (all with 1p/19qloss), 21 astrocytomas, and 5 glioblastomas. For the astrocytic lineage markers (GFAP, YKL-40, and ApoE), GFAP expression was significantly higher in the astrocytic component of oligoastrocytomas compared with the oligodendroglial part; similar patterns were detected for YKL-40 and ApoE, although the differences were not significant. GFAP, YKL-40, and ApoE reliably distinguished grade II-III oligodendrogliomas from grade II-IV astrocytomas (p < 0.0001, p = 0.002, and p < 0.0001, respectively). Among the oligodendroglial lineage markers (Olig2, Sox10, ASCL1, and NKX2-2), ASCL1 and NKX2-2 displayed significantly different immunostaining between oligodendrogliomas and astrocytomas (p = 0.017 and 0.004, respectively), but none clearly differentiated between the 2 glial populations of oligoastrocytomas. In addition to GFAP, therefore, YKL-40, ApoE, ASCL1, and NKX2-2 represent promising tumor cell markers to distinguish oligodendrogliomas from astrocytomas.     

Altered expression of immune defense genes in pilocytic astrocytomas

Pilocytic astrocytoma (WHO grade I) is a circumscribed, slowly growing, benign astrocytoma that most frequently develops in the cerebellar hemispheres and in midline structures and occurs predominantly in childhood and adolescence. In contrast to diffusely infiltrating gliomas in adults (e.g. grade II astrocytomas, oligodendrogliomas), survival of patients with pilocytic astrocytoma is excellent after surgical intervention. To search for potential molecular mechanisms underlying its benign biologic behavior, we compared gene expression profiles of pilocytic astrocytomas (8 cases) with those of normal cerebellum (4 cases), low-grade astrocytomas (WHO grade II; 15 cases), and oligodendrogliomas (WHO grade II; 17 cases) by cDNA array analysis. A number of immune system-related genes such as HLA-DRalpha, HLA-DPB1, HLA-DQB1, IgG3, IgGK, FCER1G, A2M, FCRN, IFI-56K, and DAP12 were upregulated in pilocytic astrocytomas relative to normal cerebellum, grade II astrocytomas, and oligodendrogliomas. Genes expressed at higher levels in pilocytic astrocytomas than in grade II astrocytomas and oligodendrogliomas include HLA-DRalpha, HLA-DPA1, HLA-DPB1, HLA-DQB1, A2M, TIMP1, TIMP2, CDKN1A, and SOCS3 and those expressed at lower levels include EGFR and PDGFRA. Hierarchical clustering analysis using the entire set of 1176 genes distinguished pilocytic astrocytomas from grade II astrocytomas and oligodendrogliomas. Clustering analysis using selected subgroups of genes based on their molecular functions revealed that immune system-related genes (75 genes) or cell adhesion, migration, and angiogenesis-related genes (69 genes) showed similar power to the entire gene set for separation of pilocytic astrocytomas from diffusely infiltrating low-grade gliomas. Immunohistochemistry revealed that HLA-DRalpha is expressed diffusely in neoplastic cells in pilocytic astrocytomas, whereas in oligodendrogliomas, expression was limited to scattered reactive astrocytes. These results suggest that gene expression profiles of pilocytic astrocytomas differ significantly from those of diffusely infiltrating low-grade gliomas and that their benign biologic behavior may be related to upregulation of immune defense-associated genes.     

Low grade diffuse gliomas: Shared cellular composition and morphometric differences

Low grade diffuse gliomas arising in the brain are challenging to treat because of their ability to infiltrate adjacent tissue. We attempted to clarify the cellular composition and histopathological features of low grade gliomas by utilizing morphometric and immunohistochemical analyses. Seventy‐eight cases of low grade gliomas were examined including 21 diffuse astrocytomas (DA), 36 oligodendrogliomas (OL), and 21 oligoastrocytomas (OA), based on the WHO classification system. Moreover, OL were subdivided into three types based on the morphological characteristics advocated by Daumas‐Duport et al.: OL type I, OL type II, and OL type III. The cellularity, nuclear form factor, and conditional entropy corresponding to the nuclear pleomorphism were measured in each sample by the image analysis software “Gunmetry.” Twenty‐two cases were immunohistochemically analyzed for the expression of several antigens. Morphometric data indicated that the cellularity of OL type II was significantly higher than that of DA, and that the conditional entropy of OL type III was significantly lower than that of DA. Although the results of the immunohistochemical studies were almost consistent with previous reports, there were significant differences in the expression of GFAP, nestin and p53 between DA and OL. Double immunostaining revealed that expression of Olig2 and GFAP, and Olig2 and nestin was mutually exclusive in most glioma cells. Moreover, the coexpression of nestin and GFAP occurred in DA and OA, but not in OL. We conclude that each glioma include cells expressing GFAP, cells expressing nestin, and cells expressing Olig2 in a characteristic proportion for each tumor type. We suggest that diffuse gliomas share cellular compositions in different ratios and that they can be distinguished by morphometrical analysis.     

Cyclin D1 expression in normal oligodendroglia and microglia cells: Its use in the differential diagnosis of oligodendrogliomas

Cyclin D1 regulates G1–S progression. In many carcinomas it is overexpressed and it might even correlate with prognosis. However, the amplification of CCND1 contributes to the loss of cell cycle control only in a small fraction of malignant gliomas. Cyclin D1 can be immunohistochemically demonstrated by DCS‐6 mAb. In astrocytic gliomas the fraction of tumor cells with positive nuclei is almost null in well differentiated tumors and increases with the increase of proliferation rate that occurs in anaplasia. The correct evaluation of this fraction is hindered by the positive staining of normal oligodendrocytes and microglia cells. The cyclin D1‐positive staining of normal oligodendrocytes and microglia cells has been studied in a series of 20 oligodendrogliomas, five diffuse astrocytomas and five oligoastrocytomas and in 10 samples of normal cortex and white matter, using cyclin D1 DCS‐6 mAb, Feulgen reaction and CR3.43 mAb for microglia cells. As well as microglial nuclei, the nuclei of normal oligodendrocytes of the cortex and white matter, including peri‐neuronal satellites and pericapillary cells, were immunostained by DCS‐6 mAb. In infiltrative areas of oligodendrogliomas, normal, cyclin D1‐positive oligodendrocytes and cyclin D1‐negative tumor cells coexisted. In anaplastic oligodendrogliomas, cycling tumor oligodendrocytes may regain the capacity to express cyclin D1, which is thus positive in some tumor cells. The occurrence of positive oligodendrocytes in the peripheral parts of tumors can be useful in distinguishing astrocytomas from oligoastrocytomas.     

Evidence that the central canal lining of the spinal cord contributes to oligodendrogenesis during postnatal development and adulthood in intact rats

Two waves of oligodendrogenesis in the ventricular zone of the spinal cord (SC‐VZ) during rat development, which take place between embryonic days 14 and 18 (E14–E18) and E20–E21, have been described. In the VZ of the brain, unlike the SC‐VZ, a third wave of oligodendrogenesis occurs during the first weeks of postnatal development. Using immunofluorescence staining of intact rat SC tissue, we noticed the presence of small numbers of Olig2⁺/Sox‐10⁺ cells inside the lining of the central canal (CC) during postnatal development and adulthood. Olig2⁺/Sox‐10⁺ cells appeared inside the lining of the CC shortly after birth, and their number reached a maximum of approximately 0.65 ± 0.14 cell/40‐μm section during the second postnatal week. After the latter development, the number of Olig2⁺/Sox‐10⁺ cells decreased to 0.21 ± 0.07 (P36) and 0.18 ± 0.1 cell/section (P120). At P21, Olig2⁺/Sox‐10⁺ cells inside the CC lining started to express other oligodendroglial markers such as CNPase, RIP, and APC. Olig2⁺/Sox‐10⁺ cells usually did not proliferate inside the CC lining and were only rarely found to be immunoreactive against oligodendrocyte progenitor markers such as NG2 or PDGFRα. Using 5‐bromo‐2‐deoxyuridine administration at P2, P11, P22, or P120–P125, we revealed that these cells arose in the CC lining during postnatal development and adulthood. Our findings confirmed that the CC lining is the source of a small number of cells with an oligodendroglial phenotype during postnatal development and adulthood in the SC of intact rats. J. Comp. Neurol. 522:3194–3207, 2014. © 2014 Wiley Periodicals, Inc.     

In vitro identification and functional characterization of glial precursor cells in human gliomas

Human gliomas including astrocytomas and oligodendrogliomas are defined as being composed of neoplastic astrocytes and oligodendrocytes respectively. Here, on the basis of in vitro functional assays, we show that gliomas contain a mixture of glial progenitor cells and their progeny. We have set up explant cultures from pilocytic astrocytomas, glioblastomas and oligodendrogliomas and studied antigens that characterize glial lineage, from the precursor cells (glial restricted precursors and oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells expressing the A2B5 ganglioside) to the differentiated cells (oligodendrocyte and type-1 and type-2 astrocytes). All tumoral explants contain A2B5+ cells and can generate migrating cells with distinctive functional properties according to glioma subtypes. In pilocytic astrocytomas, very few migrating cells are dividing and can differentiate in type-2 astrocytes or towards the oligodendrocyte lineage. In glioblastomas, most migrating cells are dividing, express A2B5 or glial fibrillary acid protein (GFAP) and can generate oligodendrocytes and type-1 and type-2 astrocytes in appropriate medium. Oligodendroglioma explants are made by actively dividing glial precursor cells expressing A2B5 or PSA-NCAM. Only few cells can migrate and differentiation towards oligodendrocyte lineage does not occur. Isolated A2B5+ cells from both glioblastomas and oligodendrogliomas showed similar genetic alterations as the whole tumour. Therefore, pilocytic astrocytomas contain slowly dividing oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells in keeping with their benign behaviour whereas both glioblastomas and oligodendrogliomas contain neoplastic glial restricted precursor cells. In oligodendrogliomas, these cells are trapped in undifferentiated and proliferating state. The precursor cells properties present in gliomas give new insight into their histogenesis and open up new avenues for research in the field of gliomagenesis.     

Low-grade gliomas -- current concepts

Diffuse astrocytomas, oligodendrogliomas, and oligoastrocytomas (mixed gliomas) WHO grade II, pleomorphic xanthoastrocytomas (PXAs), pilocytic astrocytomas, and subependymal giant cell astrocytomas (SEGAs) are often referred to as low-grade gliomas. WHO grade II astrocytomas, oligodendrogliomas, and mixed gliomas are characterized by their infiltrative growth, frequent tumor recurrence and a more than 50 % risk for malignant progression. In contrast, pilocytic astrocytomas and SEGAs are circumscribed tumors amenable to a (radio)surgical cure. There are few universally accepted guidelines for the treatment of low-grade gliomas. In this review, three neurosurgeons, a neurologist, a neuropathologist, and a radiation oncologist discuss some of the difficult issues surrounding the diagnosis and treatment of low-grade gliomas from their individual points of view (i. e., classification and neuropathology, MR imaging, stereotactic biopsy, microsurgery, interstitial radiotherapy/brachytherapy, radiotherapy, wait and see strategy).     

Olig2‐positive cells in glioneuronal tumors show both glial and neuronal characters: The implication of a common progenitor cell?

Glioneuronal tumors (GNTs) are rare neoplasms consisting of both glial and neuronal components. Among the GNTs, dysembryoplastic neuroepithelial tumors (DNTs), papillary glioneuronal tumors (PGNTs), and rosette‐forming glioneuronal tumors of the fourth ventricle (RGNTs) share the character of being mainly composed of small round Olig2‐positive tumor cells. Using immunohistochemistry and fluorescence in situ hybridization, we examined a series of 35 GNT cases (11 DNTs, 15 PGNTs and 9 RGNTs) on the characteristics of Olig2‐positive tumor cells. Histologically, Olig2‐positive cells showed small round forms in most GNTs; however, there were a small number of Olig2‐positive cells with neuronal morphology only in a PGNT case. These cells expressed both glial and neuronal markers by double immunostaining. With regard to labeling indices and intensity, only PGNT cells expressed neuronal markers, including α‐internexin and neurofilament. These findings also suggest that some Olig2‐positive PGNT cells may show neuronal differentiation. In GNTs, a considerable number of Olig2‐positive cells showed immunopositivity for cyclin D1 and/or platelet‐derived growth factor receptor alpha (PDGFRα), which are markers for oligodendrocyte progenitor cells. These immunostainings were particularly strong in DNTs. In RGNTs, Olig2‐positive cells formed “neurocytic rosettes”. Furthermore, they were also immunopositive for glial markers, including GFAP, PDGFRα and cyclin D1. These findings indicate the heterogeneous characteristics of Olig2‐positive cells in GNTs, and some of them also exhibited neuronal features. So it is possible that a part of Olig2‐positive GNT cells have characteristics similar to those of progenitor cells.     

Expression of retinoblastoma gene product and p21 (WAF1/Cip 1) protein in gliomas: correlations with proliferation markers, p53 expression and survival

Using immunohistochemistry we evaluated the expression of two negative regulators of the cell cycle, the retinoblastoma gene product (pRb) and the WAF1/Cip1 gene product (p21), in consecutive paraffin sections from 54 gliomas (49 astrocytomas and 5 oligodendrogliomas) and related it to clinicopathological parameters, proliferative fraction, p53 expression and survival. Survival analysis was restricted to the group of diffuse astrocytomas (48 patients). pRb expression did not correlate with histological type, grade or p53 expression, while a moderately strong correlation existed between pRb expression and the percentages of proliferating cell nuclear antigen (PCNA) and MIB-1-positive cells. In 30% of cases we observed diminished pRb expression (i.e., a low pRb/Ki-67 ratio), irrespective of grade or histological type. p21 protein was elevated in 50% of cases, especially within the higher grades. The percentage of p21-positive cells was not related to histological type or grade but correlated loosely with PCNA and pRb expression. A p53-negative/p21-negative phenotype was characteristic of oligodendrogliomas and low-grade astrocytomas, whereas the p53-positive/p21-positive, p53-positive/p21-negative and p53-negative/p21-positive phenotypes were almost equally distributed among high-grade tumors. In survival analysis (either univariate or multivariate) diminished pRb expression was not a statistically significant prognostic indicator. In contrast, p21 expression emerged as an important indicator of shortened disease-free survival, in both univariate and multivariate analyses. Moreover, the double-positive p53/p21 phenotype tended to be associated with a shorter overall survival. Our results suggest that Rb gene deregulation does not significantly affect prognosis but p21 expression may play an important role in disease-free survival of astrocytoma patients. Received: 14 August 1997 / Revised: 6 November 1997 / Accepted: 28 November 1997