[Clinicopathological characteristics and prognosis of diffuse midline gliomas with histone H3K27M mutation: an analysis of 30 cases].

Objective: To analyze the clinicopathological characteristics and prognosis of diffuse midline glioma (DMG) with H3K27M mutation. Methods: Thirty cases of DMG were collected in Guangdong Sanjiu Brain Hospital from October 2016 to May 2018. The patients' clinicopathological data including age, tumor site and histological grade, treatment and follow-up data were collected and analyzed. Results: There were 21 males and 9 females, with a mean age of 26 years (range 5-53 years). Fourteen tumors were located in thalamus, 12 in brainstem (one involved both thalamus and brainstem), and one each in hypothalamus, fourth ventricle, and sellar region, respectively. Two cases presented as diffuse intracranial lesions. Three cases (10.0%) were of WHO grade Ⅰ, 10 cases (33.3%) were grade Ⅱ, eight cases (26.7%) were grade Ⅲ, and nine cases (30.0%) were grade Ⅳ.All patients with gradeⅠ tumors were older than 20 years. Histologically, all were pilocytic astrocytoma-like. Immunohistochemical staining demonstrated that all tumors were IDH1 negative. Twenty-eight tumors showed diffuse expression of H3K27M, and two showed focal expression. Twenty-one tumors(100.0%, 21/21) showed absent expression of H3K27me3. Sixteen tumors (57.1%, 16/28) showed strongly positive expression of p53, and ATRX was negative in eight tumors (38.1%, 8/21). The Ki-67 proliferation index ranged from 5% to 40%. Eight cases (including two cases of H3K27M expression of individual cells) showed K27M mutation in H3F3A gene. Intracranial and spinal cord dissemination occurred in six cases (20.0%, 6/30). Median progression-free survival (PFS) was 9.5 months and median overall survival (OS) was 34 months. Mean PFS was 11.2 months and mean OS was 24.3 months. Compared with adults (>20 years old), children/adolescents (no more than 20 years old) had significantly shorter median OS (8 months vs. 34 months, P=0.013). There was no significant difference in PFS and OS between DMGs located in the brain stem/thalamus and other sites within midline (P>0.05). There was no significant difference in PFS and OS between WHO grade ⅠDMGs and WHO grade Ⅱ-Ⅳ DMGs (P>0.05). Conclusions: DMGs occur more commonly in children and adolescents with male predominance. DMGs present with WHO Ⅰ-Ⅳ tumors morphologically, and pilocytic astrocytoma-like lesions with WHO Ⅰ are more common in adults. Expression of H3K27M but not H3K27me3 is helpful for diagnosis of DMG. The prognosis of children/adolescents is significantly worse than that of adults, whereas histological grade and tumor location do not affect prognosis.

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Vemurafenib in pediatric patients with BRAFV600E mutated high-grade gliomas.

We present three pediatric patients with BRAFV600E mutant high-grade gliomas treated by vemurafenib on a nominative authorization level at our institution. One patient with anaplastic ganglioglioma experienced confirmed partial tumor response and significant clinical improvement and she is alive 20 months after start of treatment. A second patient with ganglioglioma responded transiently to re-introduction of vemurafenib after immunotherapy. Pharmacokinetic studies suggest that maximum concentration and exposure of vemurafenib at steady-state is dose-dependent and similar in children to that reported in adults. These cases suggest that BRAFV600 is an oncogenic driver in pediatric gliomas. Further exploration in clinical studies is ongoing.

Guggulipid and nimesulide differentially regulated inflammatory genes mRNA expressions via inhibition of NF-kB and CHOP activation in LPS-stimulated rat astrocytoma cells, C6.

Neuroinflammation is an integral part of neurodegenerative diseases. Lipo-polysacharide (LPS) induces reactive astrogliosis, the cellular manifestation of neuroinflammation, in various models of neurological diseases, but its mechanism of action is still not properly known. The effect of guggulipid and nimesulide on LPS-induced neuroinflammatory changes is also not properly understood. This work demonstrated the mechanism of actions of guggulipid and nimesulide on inflammatory genes expressions in LPS-stimulated rat astrocytoma cells, C6. We observed that LPS (10 µg/ml) treatment of rat astrocytoma cells, C6, for 24 h significantly increased intracellular Ca(2+) ion and expression of inducible nitric oxide synthase (iNOS), nuclear factor kappa-B (NF-kB), C/EBP homologous protein 10 (CHOP), c-fos, and c-jun proteins. At transcriptional stage, LPS upregulated mRNA levels of cyclooxygenase-2 and IL-6 with downregulation in IL-1α, IL-1ß, and microsomal prostaglandin E synthase-1 (mPGES-1) through activating NF-kB translocation. Treatment with guggulipid reversed these LPS-induced changes in rat astrocytoma cells. Treatment with nimesulide also attenuated LPS-induced Ca(2+) ion, iNOS, NF-kB, and c-fos expressions, but does not significantly influence CHOP, c-jun protein expressions, and mRNA levels of IL-6, IL-1α, IL-1ß, and mPGES-1 genes. In conclusion, our findings elucidated the molecular mechanism of neuroinflammation in response to LPS and its modulation by guggulipid and nimesulide in rat astrocytoma cells (C6), which suggest the use of these drugs in the treatment of neuroinflammation-associated disorders.

Tyrosine kinase expression in pediatric high grade astrocytoma.

The over-expression of several receptor tyrosine kinases in adult high grade astrocytomas (HGA) led to trials of tyrosine kinase inhibitors in these patients. Similar molecular genetic analysis of pediatric HGA is only beginning to be published. Thus it is unclear to what degree these pathways are also involved in the pediatric age group and whether they may also serve as useful therapeutic targets for children with HGAs. Here we investigated the protein expression profile of a series of pediatric HGAs. Following institutional ethical approval, clinical information and tumor samples were obtained for 42 HGA patients. Mean age at presentation was 10.1 years (range 0.13-19.3 years). OS was 12% and PFS was 3.7%. Extent of resection was associated with improved PFS (P = 0.0015) with a trend towards improved OS (P = 0.08). There was no significant effect of age or adjuvant therapy use on PFS or OS. Immunopositivity for each of the markers was as follows: p53 35%; PDGFR-alpha 45%; PDGFR-beta 31%; PTEN 67%; EGFR wild type 58%; EGFRvIII 2%. No significant effect on OS or PFS was found for any of the markers by log rank analysis. However, all long-term survivors expressed PTEN and were EGFRvIII negative. Further, there were distinct differences in protein expression between pediatric and adult HGAs suggesting that EGFR kinase inhibitors may not be beneficial for treatment of HGA in the pediatric age group and pointing to the need to study pediatric astrocytomas as distinct entities from adult astrocytomas.

Ethanol-induced modulation of inducible nitric-oxide synthase activity in human A172 astrocytoma cells.

BACKGROUND: Glial cells are critical in the functioning of the central nervous system (CNS), including responsiveness to injury and immunocompetence. The immune and inflammatory response involves the inducible form of nitric-oxide synthase (iNOS), and subsequent nitric oxide (NO) production. Previously, we have demonstrated that ethanol inhibits cytokine-induced iNOS expression and activity in rat glial cells. Evidence of ethanol-induced effects on iNOS in human glial cells is nonexistent. Herein, the conditions necessary for significant iNOS induction in human A172 astrocytoma cells have been characterized, and subsequently, the effects of ethanol on iNOS expression have been investigated. METHODS: A172 cells were analyzed immunohistochemically for the astrocyte markers, glial fibrillary acidic protein (GFAP) and S-100beta. The ability of A172 cells to express iNOS was assessed by stimulating cells with interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), bacterial lipopolysaccharide (LPS), L-arginine, and tetrahydrobiopterin (BH4) in various combinations. Following stimulation, iNOS induction was monitored via measurement of nitrite production and in vitro iNOS enzyme activity. Time-course (6-24 hr) studies assessed the effects of ethanol (50-200 mM) on iNOS induction. RESULTS: Immunohistochemistry analysis confirmed that A172 cells were phenotypically, astrocytic. Induction of nitrite production by a cytomix [IFNgamma (100 ng/ml) + TNFalpha (30 ng/ml) + IL-1beta (5 ng/ml)] was differentially enhanced by exposure to supplemental factors including LPS, L-arginine, and BH4. Nitrite production was greatest over the initial 24 hr of stimulation with iNOS enzyme activity peaking at 12 hr. Acute (6-24 hr) exposure of activated cells to 50 mM ethanol enhanced iNOS activity recovered from the cytosol, whereas 200 mM ethanol decreased it. Ethanol had no direct effect on the catalytic activity of the enzyme. CONCLUSIONS: The present study is the first published report of ethanol-induced modulation of iNOS expression in human glial cells. The data suggest that ethanol is influencing iNOS enzyme levels most profoundly. Altered astrocyte function may be a point of ethanol-induced perturbation in CNS immune function. These findings should lend insight into the role of ethanol on human CNS immunity and brain injury.

Expression of cyclooxygenase 2 (COX-2) in human glioma and in vitro inhibition by a specific COX-2 inhibitor, NS-398.

The up-regulation of cyclooxygenase 2 (COX-2) expression is a frequent occurrence in a variety of different tumors. In this study, COX-2 protein expression was investigated in 50 glioma and 3 normal brain specimens by immunohistochemistry. Expression of COX-2 protein was observed in all normal brain and glioma specimens by immunohistochemistry, regardless of histological grade. The immunoreactive score was significantly higher in high-grade glioma than low-grade glioma and normal brain specimens. For a subset of these tumors (nine gliomas and three normal brain), Western blot analysis was also performed. COX-2 protein was detected in all specimens by Western blot analysis. The effect of the specific COX-2 inhibitor NS-398 on monolayer cell cultures and three-dimensional glioma spheroids was investigated using U-87MG and U-251MG human glioblastoma cell lines. The proliferation rate was assessed in monolayer cultures. In addition, a growth assay, a migration assay, an apoptosis assay, and a tumor invasion assay were performed in a three-dimensional spheroid culture system. NS-398 was able to reduce the proliferation of monolayer cell cultures, as well as the growth of spheroids and tumor cell migration, in a dose-dependent manner. There was also a moderate increase in the number of apoptotic cells in the treated spheroids. NS-398 did not have an inhibitory effect on tumor invasion in the coculture spheroid system. Our study provides evidence that COX-2 is up-regulated in the majority of high-grade gliomas and that a potential role of COX-2 inhibitors as an adjuvant therapy for brain tumors may exist.

Cytokine-induced transcription of protein-tyrosine-phosphatases in human astrocytoma cells.

Interleukin-1 (IL-1) and Tumor Necrosis Factor-a (TNFalpha) are potent mediators of inflammatory reactions in the brain. Although much is known about the effects of IL-1 on expression of secretory proteins, few studies have addressed the question of a selective, IL-1-dependent expression of genes involved in neuromodulatory effects of inflammation. Protein-tyrosine-phosphatases (PTP's) have been shown to regulate signal transduction and adhesion processes in the developing nervous system. They are candidates for inflammation-induced neuromodulation. Therefore, we investigated if IL-1 regulates expression of PTP's. We applied a DNA-fingerprinting method based on the PCR-amplification of conserved domains of gene families and observed IL-1-dependent induction of two PTP's, cytoplasmic PTPvarepsilon and receptor-PTPgamma, RPTPgamma, in human U373-MG astrocytoma cells. Using Northern blot analysis, we confirmed this result and also show that in addition to IL-1, TNFalpha but not IL-6 induces the transcription of cytoplasmic PTPvarepsilon and RPTPgamma in human astrocytoma cells. Given the important role for PTP's in neuromodulatory aspects such as axonal guidance and neurite outgrowth, cytokine-induced induction of PTP's may play an important pathenogenic role in the development of chronic inflammatory diseases in the brain.