Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease.

Little is known about the genetic regulation of medulloblastoma dissemination, but metastatic medulloblastoma is highly associated with poor outcome. We obtained expression profiles of 23 primary medulloblastomas clinically designated as either metastatic (M+) or non-metastatic (M0) and identified 85 genes whose expression differed significantly between classes. Using a class prediction algorithm based on these genes and a leave-one-out approach, we assigned sample class to these tumors (M+ or M0) with 72% accuracy and to four additional independent tumors with 100% accuracy. We also assigned the metastatic medulloblastoma cell line Daoy to the metastatic class. Notably, platelet-derived growth factor receptor alpha (PDGFRA) and members of the downstream RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway are upregulated in M+ tumors. Immunohistochemical validation on an independent set of tumors shows significant overexpression of PDGFRA in M+ tumors compared to M0 tumors. Using in vitro assays, we show that platelet-derived growth factor alpha (PDGFA) enhances medulloblastoma migration and increases downstream MAP2K1 (MEK1), MAP2K2 (MEK2), MAPK1 (p42 MAPK) and MAPK3 (p44 MAPK) phosphorylation in a dose-dependent manner. Neutralizing antibodies to PDGFRA blocks MAP2K1, MAP2K2 and MAPK1/3 phosphorylation, whereas U0126, a highly specific inhibitor of MAP2K1 and MAP2K2, also blocks MAPK1/3. Both inhibit migration and prevent PDGFA-stimulated migration. These results provide the first insight into the genetic regulation of medulloblastoma metastasis and are the first to suggest a role for PDGFRA and the RAS/MAPK signaling pathway in medulloblastoma metastasis. Inhibitors of PDGFRA and RAS proteins should therefore be considered for investigation as possible novel therapeutic strategies against medulloblastoma.

The Ewing tumor family of peripheral primitive neuroectodermal tumors expresses human gastrin-releasing peptide.

The Ewing tumor family of peripheral primitive neuroectodermal tumors (pPNETs) are characterized by chromosomal translocations leading to EWS-ETS gene fusions. These hybrid genes express chimeric proteins that are thought to act as aberrant transcription factors. We therefore used differential display-PCR to compare gene expression patterns in pPNET cell lines with those of other small round cell tumors (SRCTs) of childhood. This technique detected differential expression of sequences corresponding to human gastrin-releasing peptide (GRP) in pPNET cell lines but not in other SRCT cell lines. Subsequent Northern and reverse transcription-PCR analysis of SRCT cell lines confirmed GRP positivity in all pPNET lines tested. Of primary tumors tested by reverse transcription-PCR, GRP expression was found in 7 (44%) of 16 pPNETs but in no other primary SRCTs examined. Expression of the GRP receptor gene was demonstrable in 55% of pPNET cell lines and 25% of primary pPNET tumors but also in several other SRCTs. Radioimmunoassays and immunohistochemistry confirmed expression of bioactive GRP peptide in pPNET cell lines and primary tumors, respectively. Moreover, in vitro growth of a pPNET cell line was slowed by treatment with a GRP receptor antagonist and accelerated by a GRP receptor agonist. GRP is a known autocrine growth factor in small cell lung cancer and other neuroendocrine tumors. Its expression in pPNETs provides further evidence for a neuroectodermal histogenesis of these tumors and suggests that autocrine growth of this family of tumors may be at least partially regulated by GRP.

Detection of brain tumor invasion and micrometastasis in vivo by expression of enhanced green fluorescent protein.

OBJECTIVE: To determine whether fluorescence from human brain tumor cells transfected with the enhanced green fluorescent protein (EGFP) gene in vitro and xenotransplanted into the brain of nude mice would permit the detection of brain tumor invasion and metastasis in vivo. METHODS: Daoy medulloblastoma cells were transfected with a long terminal repeat-based retroviral vector containing the EGFP gene. Stable EGFP-expressing clones were isolated and stereotactically injected into the frontal cortex of nude mice. Four weeks later, whole brain sections were examined using fluorescence microscopy, immunohistochemistry, and routine hematoxylin and eosin staining for the visualization and detection of tumor cell invasion and metastasis. RESULTS: We demonstrate that EGFP-transduced Daoy cells maintain stable high-level EGFP expression in the central nervous system during their growth in vivo. EGFP fluorescence clearly demarcated the primary tumor margins and readily allowed for the visualization of distant micrometastases and local invasion on the single-cell level. Small metastatic and locally invasive foci, including those immediately adjacent to the tumor's leading invasive edge, were virtually undetectable by routine hematoxylin and eosin staining and immunohistochemistry. EGFP expression also persisted in vitro after cell reculture from brain tissue extracts. CONCLUSION: We show, for the first time, that EGFP-transduced human brain tumor cells can be visualized by fluorescence microscopy after intracerebral implantation. This method is superior to routine hematoxylin and eosin staining and immunohistochemistry for the detection and study of physiologically relevant patterns of brain tumor invasion and metastasis in vivo.

Preferential susceptibility of brain tumors to the antiangiogenic effects of an alpha(v) integrin antagonist.

OBJECTIVE: Brain tumors are highly angiogenic, and their growth and spread depend on the generation of new blood vessels. We examined the effect of the cyclic peptide antagonist pentapeptide EMD 121974, an antiangiogenic agent, on orthotopic and heterotopic brain tumor growth. METHODS: The human brain tumor cell lines DAOY (medulloblastoma) and U87 MG (glioblastoma) were injected into either the forebrain (orthotopic) or the subcutis (heterotopic) of nude mice, and daily systemic treatment with the active peptide was initiated after tumors were established. RESULTS: All control animals with orthotopic brain tumors and that received the inactive peptide EMD 135981 daily died as a result of tumor progression within 4 to 6 weeks; tumors measured 3 to 5 mm in diameter. In contrast, mice with orthotopic tumors that were treated daily with the active peptide survived for more than 16 weeks, and histological examination of the brains after 4, 8, and 12 weeks showed either no tumors or microscopic residual tumors. The growth of these brain tumor cells injected simultaneously or separately into the subcutis of nude mice (heterotopic model) was not affected by the active peptide, suggesting that the brain environment is a critical determinant of brain tumor susceptibility to growth inhibition by this pentapeptide. CONCLUSION: The cyclic pentapeptide EMD 121974 may become a treatment option specific to brain tumors. Because of its antiangiogenic effect, its use may be especially indicated after tumors are removed surgically.

Antisense to integrin alpha v inhibits growth and induces apoptosis in medulloblastoma cells.

BACKGROUND: Integrin alpha v promotes brain microvessel endothelium survival, yet its role in brain tumor cells is unknown. MATERIALS AND METHODS: Alpha v synthesis in medulloblastoma cells Daoy, D341Med, and D283Med, was inhibited with antisense oligonucleotides (ASODN) to test the effect on growth and survivaL RESULTS: ASODN reduced alpha v surface expression 75% in a dose- and time-dependent manner (2 microM, 72 hours). Alpha v-deficient cells grown with vitronectin demonstrated reduced cell spreading, G0-G1 growth arrest, decreased proliferation and increased apoptosis compared to controls or alpha v-deficient cells grown with collagen. Furthermore, insulin-like growth factor-I (IGF-I) suboptimally stimulated proliferation and survival of alpha v-deficient cells, suggesting that alpha v-IGF-I interactions potentiate medulloblastoma growth. Finally, treatment with alpha v-blocking antibody induced caspase-8 and caspase-3 expression, while apoptosis of alpha v-deficient cells was associated only with increased caspase-3. CONCLUSION: Alpha v integrin supports medulloblastoma growth by activating adhesion-dependent and -independent survival pathways and thus may serve as a novel therapeutic target in this tumor.

Treatment of high-grade glioma in children and adolescents.

Pediatric high-grade gliomas (HGGs)--including glioblastoma multiforme, anaplastic astrocytoma, and diffuse intrinsic pontine glioma--are difficult to treat and are associated with an extremely poor prognosis. There are no effective chemotherapeutic regimens for the treatment of pediatric HGG, but many new treatment options are in active investigation. There are crucial molecular differences between adult and pediatric HGG such that results from adult clinical trials cannot simply be extrapolated to children. Molecular markers overexpressed in pediatric HGG include PDGFRα and P53. Amplification of EGFR is observed, but to a lesser degree than in adult HGG. Potential molecular targets and new therapies in development for pediatric HGG are described in this review. Research into bevacizumab in pediatric HGG indicates that its activity is less than that observed in adult HGG. Similarly, tipifarnib was found to have minimal activity in pediatric HGG, whereas gefitinib has shown greater effects. After promising phase I findings in children with primary CNS tumors, the integrin inhibitor cilengitide is being investigated in a phase II trial in pediatric HGG. Studies are also ongoing in pediatric HGG with 2 EGFR inhibitors: cetuximab and nimotuzumab. Other novel treatment modalities under investigation include dendritic cell-based vaccinations, boron neutron capture therapy, and telomerase inhibition. While the results of these trials are keenly awaited, the current belief is that multimodal therapy holds the greatest promise. Research efforts should be directed toward building multitherapeutic regimens that are well tolerated and that offer the greatest antitumor activity in the setting of pediatric HGG.

CNS germ cell tumor (CNSGCT) of childhood: presentation and delayed diagnosis.

OBJECTIVE: To describe the relationship between symptomatology and time to diagnosis of an institutional series of patients with CNS germ cell tumor (CNSGCT) over a 16-year period. METHODS: Thirty consecutive patients newly diagnosed with CNSGCT (mean age 10.9 years; range 6 to 17 years; 70% boys) were evaluated at our institution between 1990 and 2006. RESULTS: Duration of symptoms prior to diagnosis ranged from 5 days to 3 years (mean 8.4 months). Tumor location included pineal (14), suprasellar (8), pineal/suprasellar (3), pineal/thalamic (4), and basal ganglionic/thalamic (3). Five patients had disseminated disease at the time of diagnosis. Features including headache, nausea, vomiting, and visual changes led to earlier diagnosis. Symptoms including movement disorders, enuresis, anorexia, and psychiatric complaints delayed diagnosis in 9 of 30 patients, diagnosed 7 months to 3 years (mean 22.3 months) from symptom onset. In 7 of 9 patients with delayed diagnosis, enuresis was present. Seventeen of 30 patients had signs of endocrine dysfunction at presentation that included diabetes insipidus (4), hypothyroidism (8), and growth hormone deficiency (4). Ophthalmologic findings of decreased visual acuity, visual field deficits, or ocular abnormalities were present in 13 patients. Duration of symptoms did not correlate with tumor subtype or event-free survival. In three patients with basal ganglionic/temporal lobe, thalamic, or pineal/suprasellar signal abnormalities on MRI, neuroradiographic diagnosis was difficult. CONCLUSIONS: Diagnosis of CNS germ cell tumor is often delayed, and presentation may include movement disorders or mimic psychiatric disease. MRI interpretation can be challenging and may require serum/CSF markers and biopsy for diagnosis.

Urokinase induces receptor mediated brain tumor cell migration and invasion.

The plasminogen activation (PA) system plays an important role in tumor invasion by initiating pericellular proteolysis of the extracellular matrix (ECM) and inducing cell migration. Malignant brain tumors overexpress PA members and characteristically invade by migrating on ECM-producing white matter tracts and blood vessel walls. To determine whether urokinase-type plasminogen activator (uPA) and its receptor (uPAR) directly modulate the migration of brain tumor cells, we examined six human brain tumor cell lines, 2 astrocytomas (SW1088, SW1783), 2 medullobastomas (Daoy, D341Med), and 2 glioblastomas (U87MG, U118MG), for their surface uPAR expression, endogenous PA activity, and functional proteolytic activity by an ECM-degradation assay. Migration on Transwell membranes and invasion of Matrigel was then tested by pre-incubating the cells with increasing concentrations of either uPA, the proteolytically inactive amino-terminal fragment (ATF) of uPA, or the uPAR cleaving enzyme, phosphatidylinositol-specific phospholipase C (PI-PLC). All of the cell lines, except D341Med, express surface uPAR protein and uPA activity. High levels of uPAR and uPA activity correlated with cellular degradation of ECM, cell migration, and Matrigel invasion. Cell migration and invasion were enhanced by uPA or ATF in a dose dependent manner, while PI-PLC treatment abolished the uPA effect and inhibited migration and invasion. We conclude that ligation of uPAR by uPA directly induces brain tumor cell migration, independent of uPA-mediated proteolysis; and in concert with ECM degradation, markedly enhances invasion. Conversely, removing membrane bound uPAR from the surface of the cells studied inhibited their ability to migrate and invade even in the presence of proteolytically active uPA.

Determination of urinary mandelic and phenylglyoxylic acids in styrene exposed workers and a control population.

Styrene is rapidly metabolized in humans to mandelic () and phenylglyoxylic acids (P) which are excreted in urine. The present study investigates a gas chromatographic technique for measuring urinary concentrations of MA and PGA of workers exposed to styrene, compares the urinary concentrations of metabolites with time-weighted average air exposures to styrene and determines the levels of these metabolites in a population of workers not exposed to styrene. Post-shift urine specimens were obtained from a group of workers exposed to styrene in the reinforced plastic industry and from a control group. High positive correlation was found between post-shift urinary concentrations of metabolites and 8-hour TWA styrene exposure. Both MA and total metabolites (MA + PGA) gave correlation coefficient values of 0.96, p less than 0.0001. The mean MA excretion for the control groups was 6 mg/L. Determination of the concentration of these metabolites in a post-shift urine provides an effective means of estimating and monitoring human exposure to styrene.