Immunohistochemical analyses of neural stem cell lineage markers in normal feline brains and glial tumors.

Neural stem cell (NSC) lineage cells have not been fully identified in feline brains, and the NSC-like nature of feline glial tumors has not been determined. In this study, 6 normal cat brains (3 newborn and 3 older cats) and 13 feline glial tumors were analyzed using immunohistochemical NSC lineage markers. The feline glial tumors were subjected to immunohistochemical scoring followed by hierarchical cluster analysis. In newborn brains, glial acidic fibrillary protein (GFAP)/nestin/sex-determining region Y-box transcription factor 2 (SOX2)-immunopositive NSCs, SOX2-immunopositive intermediate progenitor cells, oligodendrocyte transcription factor 2 (OLIG2)/platelet-derived growth factor receptor-α (PDGFR-α)-immunopositive oligodendrocyte precursor cells (OPCs), OLIG2/GFAP-immunopositive immature astrocytes, and neuronal nuclear (NeuN)/ß-3 tubulin-immunopositive mature neuronal cells were observed. The apical membrane of NSCs was also immunopositive for Na+/H+ exchanger regulatory factor 1 (NHERF1). In mature brains, the NSC lineage cells were similar to those of the newborn brains. A total of 13 glial tumors consisted of 2 oligodendrogliomas, 4 astrocytomas, 3 subependymomas, and 4 ependymomas. Astrocytomas, subependymomas, and ependymomas were immunopositive for GFAP, nestin, and SOX2. Subependymomas and ependymomas showed dot-like or apical membrane immunolabeling for NHERF1, respectively. Astrocytomas were immunopositive for OLIG2. Oligodendrogliomas and subependymomas were immunopositive for OLIG2 and PDGFR-α. Feline glial tumors also showed variable immunolabeling for ß-3 tubulin, NeuN, and synaptophysin. Based on these results, feline astrocytomas, subependymomas, and ependymomas appear to have an NSC-like immunophenotype. In addition, astrocytomas, subependymomas, and ependymomas have the characteristics of glial, oligodendrocyte precursor, and ependymal cells, respectively. Feline oligodendrogliomas likely have an OPC-like immunophenotype. In addition, feline glial tumors may have multipotential stemness for differentiation into neuronal cells. These preliminary results should be validated by gene expression analyses in future studies with larger case numbers.

Water Channels Aquaporin 4 and -1 Expression in Subependymoma Depends on the Localization of the Tumors.

BACKGROUND: We analyzed aquaporin 4 and -1 expression in subependymomas, benign and slow growing brain tumors WHO grade I. Ten subependymoma cases were investigated, five of the fossa inferior and five of the fossa superior. METHODS AND RESULTS: Using immunohistochemistry, we observed different aquaporin expression patterns depending on localization: aquaporin 4 and -1 were detected in infratentorial subependymomas in the entire tumor tissue. In contrast, supratentorial subependymomas revealed aquaporin 4 and -1 expression only in border areas of the tumor. PCR analyses however showed no difference in aquaporin 4 expression between all subependymomas independent of localization but at higher levels than in normal brain. In contrast, aquaporin 1 RNA levels were found to be higher only in infratentorial samples compared to supratentorial and normal brain samples. The reason for the different distribution pattern of aquaporin 4 in subependymomas still remains unclear. On the cellular level, aquaporin 4 was redistributed on the surface of the tumor cells, and in freeze fracture replicas no orthogonal arrays of particles were found. This was similar to our previous findings in malignant glioblastomas. From these studies, we know that extracellular matrix molecules within the tumor like agrin and its receptor alpha-dystroglycan are involved in forming orthogonal arrays of particles. In subependymomas neither agrin nor alpha-dystroglycan were detected around blood vessels. CONCLUSIONS: Taken together, we show in this study that in the benign subependymomas aquaporins 1 and 4 are dramatically redistributed and upregulated. We speculate that extracellular environments of infra- and supratentorial subependymomas are different and lead to different distribution patterns of aquaporin 4 and -1.

Therapeutic targets in subependymoma.

Subependymomas are usually treated with surgical resection; however, no standard, defined alternative medical therapy is recommended for patients who are not surgical candidates, owing to a paucity of molecular, immunological, and genetic characterization. To address this, an ex vivo functional analysis of the immune microenvironment in subependymoma was conducted, a subependymoma cytokine/chemokine microarray was constructed for the evaluation of operational immune and molecular pathways, and a subependymoma cell line was derived and used to test a variety of cytotoxic agents that target operational pathways identified in subependymoma. We found that immune effectors are detectable within the microenvironment of subependymoma; however, marked immune suppression is not observed. The subependymoma tissue microarrays demonstrated tumor expression of p53, MDM2, HIF-1α, topoisomerase II-ß, p-STAT3, and nucleolin, but not EGFRvIII, EphA2, IL-13RA2, CMV, CTLA-4, FoxP3, PD-1, PD-L1, EGFR, PDGF-α, PDGF-ß, PDGFR-α, PDGFR-ß, PTEN, IGFBP2, PI3K, MDM4, IDH1, mTOR, or Jak2. A topoisomerase inhibitor (WP744, IC50=0.83 µM) and a p-STAT3/HIF-1α inhibitor (WP1066, IC50=3.15 µM) demonstrated a growth inhibition of the subependymoma cell proliferation. Cumulatively, these data suggest that those agents that interfere with oncogenes operational in subependymoma may have clinical impact.

Prognostic relevance of global histone 3 lysine 9 acetylation in ependymal tumors.

OBJECT: Ependymal tumors are highly variable in clinical and molecular behavior and affect both children and adults. Regarding the paucity of appropriate experimental models, the underlying molecular mechanisms of their behavioral variability are poorly understood. Considering the increasing evidence of epigenetic changes in various tumors, in addition to the preclinical success of epigenetic-based therapeutics in tumors of the CNS, epigenetic study of ependymal tumors is warranted. METHODS: Using immunohistochemistry, the authors investigated the patterns of global acetylation of lysine position 9 of histone 3 (H3K9Ac), an epigenetic marker of active gene transcription, in 85 ependymal tumors with various WHO grades and clinicopathological characteristics. RESULTS: Most of the nuclei in all ependymal tumors were H3K9Ac negative (mean ± SD 65.9% ± 26.5 vs 34.1% ± 26.5% positive, p < 0.0001). Subependymomas had more H3K9Ac-positive nuclei (67.2% ± 10.2%) than myxopapillary ependymomas, ependymomas, and anaplastic ependymomas (p < 0.05). Additionally, intracranial parenchymal tumors had significantly fewer H3K9Ac-positive nuclei (13.1% ± 21.9%) than tumors of other CNS localizations (p < 0.001), and supratentorial ventricular tumors had the highest number of H3K9Ac-positive nuclei (66.4% ± 11.8%) among CNS ependymal tumors (p < 0.0001). The H3K9Ac pattern in ependymal tumors also revealed prognostic significance such that tumors with less than 20% acetylated nuclei had a higher probability of recurrence than tumors with 20% or more acetylated nuclei (p = 0.0327), and recurrent tumors had significantly fewer H3K9Ac-positive nuclei than primary ones (16% ± 22.5% vs. 38% ± 25.8%; p < 0.0001). However, the effect of tumor location on survival of patients was nonsignificant in a multivariate survival analysis, and H3K9 acetylation levels of tumors contributed independently to the survival of patients. In addition, ependymal tumors with more than or equal to 20% H3K9 acetylated cells had lower MIB-1 expression than those with less than 20% H3K9 acetylated cells (p < 0.01). CONCLUSIONS: Global H3K9Ac contributes independently to the prognosis of patients with ependymal tumors such that tumors with lower H3K9Ac values have a higher probability of recurrence and are more proliferative. Additionally, subependymomas have a higher H3K9Ac profile than other ependymal tumor subclasses, underlining their benign clinical behavior.

Subependymomas of the lateral ventricle: tumor recurrence correlated with increased Ki-67 labeling index.

Subependymomas of the lateral ventricles are rare tumors. We present two patients with subependymomas of the lateral ventricle, who underwent gross total resection of the tumor via transcallosal approach. The patient, with increased Ki-67 labeling index had recurrence of tumor two years after the initial operation. We emphasize at the risk of recurrence which is probably correlated with Ki-67 labeling index.

Cellular localization of metabotropic glutamate receptors in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder associated with cortical malformations (cortical tubers) and the development of glial tumors (subependymal giant-cell tumors, SGCTs). Expression of metabotropic glutamate receptor (mGluR) subtypes is developmentally regulated and several studies suggest an involvement of mGluR-mediated glutamate signaling in the regulation of proliferation and survival of neural stem-progenitor cells, as well as in the control of tumor growth. In the present study, we have investigated the expression and cell-specific distribution of group I (mGluR1, mGluR5), group II (mGluR2/3) and group III (mGluR4 and mGluR8) mGluR subtypes in human TSC specimens of both cortical tubers and SGCTs, using immunocytochemistry. Strong group I mGluR immunoreactivity (IR) was observed in the large majority of TSC specimens in dysplastic neurons and in giant cells within cortical tubers, as well as in tumor cells within SGCTs. In particular mGluR5 appeared to be most frequently expressed, whereas mGluR1alpha was detected in a subpopulation of neurons and giant cells. Cells expressing mGluR1alpha and mGluR5, demonstrate IR for phospho-S6 ribosomal protein (PS6), which is a marker of the mammalian target of rapamycin (mTOR) pathway activation. Group II and particularly group III mGluR IR was less frequently observed than group I mGluRs in dysplastic neurons and giant cells of tubers and tumor cells of SGCTs. Reactive astrocytes were mainly stained with mGluR5 and mGluR2/3. These findings expand our knowledge concerning the cellular phenotype in cortical tubers and in SGCTs and highlight the role of group I mGluRs as important mediators of glutamate signaling in TSC brain lesions. Individual mGluR subtypes may represent potential pharmacological targets for the treatment of the neurological manifestations associated with TSC brain lesions.

Genome-wide analysis of subependymomas shows underlying chromosomal copy number changes involving chromosomes 6, 7, 8 and 14 in a proportion of cases.

Subependymomas (SE) are slow-growing brain tumors that tend to occur within the ventricles of middle-aged and elderly adults. The World Health Organization classifies these tumors within the ependymoma group. Previous limited analysis of this tumor type had not revealed significant underlying cytogenetic abnormalities. We have used microarray comparative genomic hybridization to study a series of SE (n = 12). A whole-genome array at 0.97-Mb resolution showed copy number abnormalities in five of 12 cases (42%). Two cases (17%) showed regions of loss on chromosome 6. More detailed analysis of all cases using a chromosome 6 tile-path array confirmed the presence of overlapping regions of loss in only these two cases. One of these cases also showed trisomy chromosome 7. Monosomy of chromosome 8 was seen in a further two cases (17%), and a partial loss on chromosome 14 was observed in one additional case. This is the first array-based, genome-wide study of SE. The observation that five of 12 cases examined (42%) at 0.97-Mb resolution showed chromosomal copy number abnormalities is a novel finding in this tumor type.

Immunohistochemical study of central neurocytoma, subependymoma, and subependymal giant cell astrocytoma.

For investigation of histogenesis of central neurocytomas (CNs), subependymoma (SEs), subependymal giant cell astrocytomas (SEGAs), we studied expression of various neuronal and glial biomarkers by immunohistochemical (IHC) study and reverse transcriptase-polymerase chain reaction (RT-PCR). The materials for IHC were paraffin section of seven CNs, three SEs, and eight SEGAs and those for RT-PCR were frozen tissues of seven CNs, three SEs, and five SEGAs. Control group was five ependymomas (EPs) and four pilocytic astrocytomas (PAs). The neuronal biomarkers included nestin, chromogranin A (chrA), synaptophysin (SNP), neuronal cell adhesion molecule (NCAM), neuron specific enolase (NSE), neuronal nuclear antigen (NeuN), neurofilament (NF) and the glial marker was GFAP. CNs expressed all neuronal markers except NF (0%), SNP (100%), NCAM (100%), NSE (100%), NeuN (100%), nestin (29%) and chrA (43%), but GFAP expression was found only in one case (14%). SEGA coexpressed several neuronal markers and a glial marker; NeuN (100%), NSE (88%), NCAM (63%), nestin (100%), SNP (weakly and focally, 100%), and GFAP (100%), however, other neuronal markers including chrA, SNP and NF were all negative. SE expressed nonspecific neuronal markers (NCAM (100%) and NSE (100%)) which showed weak intensity and a GFAP (100%), but not nestin. Among control cases of EPs and PAs, no one case expressed neuronal markers except nonspecific neuronal marker of NCAM, but robustly expressed GFAP. RT-PCR product of nestin was expressed in 29% of CNs (2/7cases), 60% of SEGAs (3/5 cases), 100% of SEs (3/3 cases), 80% of EPs (4/5 cases), and 25% of PAs (1/4 cases). Conclusively, coexpression of neuronal and glial markers and expression of nestin in CNs, SEGAs and SEs suggested the origin of these tumor cells might be the stem cells being able to differentiate into both neuronal and glial phenotypes. But CNs might be originated from rather neuronally committed stem cells and SEs from rather glially committed stem cells.

Supratentorial extraventricular ependymal neoplasms: a clinicopathologic study of 32 patients.

BACKGROUND: Published research on the clinicopathologic features of extraventricular ependymal neoplasms of the cerebral hemispheres has been scant. METHODS: Thirty-two archival cases were studied to investigate the prognostic impact of clinicopathologic parameters including flow cytometry, the proliferation (Ki-67) labeling index, and p53 expression. RESULTS: Among these 32 cases were 2 subependymomas, 19 ependymomas, and 11 anaplastic ependymomas. No significant gender predilection was observed, and 45% of patients were in their second or third decade of life. The left cerebral hemisphere was 1.5 times more commonly involved. On available imaging studies, lesions were often cystic, with or without a mural nodule. Tumors expressed glial fibrillary acidic protein (87%), S-100 protein (77%), cytokeratin (43%), and epithelial membrane antigen (17%). Ki-67 proliferation index paralleled tumor grade. Immunoreactivity for p53 protein was observed in the 2 cases of subependymoma, in 10 of 11 anaplastic ependymomas, and in 6 of 17 ependymomas. Flow cytometry performed in 27 tumors revealed diploidy in 20 cases and aneuploidy in 4 cases (3 anaplastic and 1 classic ependymomas), with S-phase fraction ranging from 0.2-9.7. Eleven subjects were additionally treated with radiotherapy, and 3 with chemotherapy. Follow up was available in 25 (78%) patients. CONCLUSIONS: The results of the current study suggest that there is no significant relation between histopathology, Ki-67 proliferation index, p53 immunolabeling, tumor ploidy, and biologic behavior.

Subependymal giant cell astrocytoma: a clinicopathological study of 23 cases with special emphasis on proliferative markers and expression of p53 and retinoblastoma gene proteins.

AIMS: To gain a better insight into the biological behaviour of subependymal giant cell astrocytoma (SEGA), tumour suppressor gene protein expression and various proliferative indices were studied in these tumours and correlated with histological features and clinical outcome. METHODS: We studied 23 cases of SEGA, 19 from our own Institute and four from the National Institute of Mental Health and Neurological Sciences (NIMHANS), Bangalore, India. Immunohistochemical staining for various glial and neuronal markers, proliferative markers (MIB-1, Topoisomerase II alpha PCNA) and tumour suppressor gene protein expression of p53 and retinoblastoma (Rb) were performed. RESULTS: Nineteen cases of SEGA were collected over a period of 23 years (January 1978-December 2001), which accounted for 0.16% of all intracranial tumours and 0.51% of all gliomas reported at this centre. Ages ranged from 4 to 37 years (mean 13.2 years) with a male preponderance. Nine of the 23 cases were associated with tuberous sclerosis (TS), six at the time of diagnosis, while three developed TS during the follow-up period. Treatment consisted of surgical resection (total in nine cases and subtotal in 14 cases) followed by radiotherapy in seven cases. Except for two patients who died in the immediate post-operative period of surgical complications, the remaining patients were all alive in the follow-up period (mean 37.1 months). One patient experienced recurrence 22 years after the first surgery and a second patient after 2 years. Necrosis and/or mitoses were observed in five cases. Immunohistochemically, tumours were positive for both glial and neuronal markers. Interspersed inflammatory cells were a mixture of mast cells and lymphocytes of T immunophenotype. The MIB-1 labelling index (LI) ranged from 0 to 8% (mean 3.0%), topoisomerase II alpha (topo II alpha) LI from 0 to 9.5% (mean 2.9%) and PCNA LI from 10 to 59% (mean 32.5%). The difference in the labelling indices of tumours with and without mitoses and/or necrosis was not statistically significant. None of the tumours revealed loss of Rb gene protein expression. p53 immunopositivity was seen in 14 cases (labelling indices ranged from 1 to 7.3% with a mean of 2.4%). The correlation between the MIB-1 LI and topo II alpha LI, and topo II alpha LI and PCNA LI was significant (P<0.05) but not so with other parameters like p53 protein expression, duration of survival and morphological features such as mitoses and necrosis. CONCLUSIONS: SEGAs are rare intraventricular tumours associated with TS and express both neuronal and glial markers. They have a low proliferative potential. Mitoses and necrosis are not associated with a worse prognosis. In view of the low proliferative indices and long survival of these patients without recurrence, the role of post-operative radiotherapy is questionable. These patients should be followed up closely as many of them develop stigmata of tuberous sclerosis at a later stage.

Epidemiology and pathology of intraventricular tumors.

Tumors that primarily or exclusively involve the ventricular system constitute a rare and heterogeneous group. Certain histologic tumor types predominantly occur in children, whereas others are more common in adults. Tumor location provides additional clues to correct diagnosis. When used in conjunction with clinical and radiologic data, histopathologic features can distinguish among this wide range of possibilities to provide the correct diagnosis for optimal patient management.

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

Magnetic resonance spectroscopy study of low grade extra and intracerebral human neoplasms.

In this study we report an in vitro 1H magnetic resonance spectroscopy (MRS) characterization of water soluble metabolites obtained from 17 low grade extra and intracerebral human tumors (8 meningiomas, 5 oligodendrogliomas, 2 subependimomas and 2 ependimomas). In addition, the in vivo localized 1H MRS results for 4 of the meningiomas are reported. The main metabolic features characterizing low grade tumors were investigated. Meningiomas are characterized by high Cho/Cr and Ala/Cr ratios; in many cases both in the in vitro and the in vivo spectra these ratios cannot be evaluated, due to the absence of the Cr metabolite. Low grade oligodendrogliomas are characterized by low Cho/Cr ratios. High amounts of myo-inositol are found in the spectra of ependimomas and subependimomas which are distinguished by different Cho/Cr ratio values and by a different Ala content.