Methylome Alterations “Mark” New Therapeutic Opportunities in Glioblastoma

In this issue of Cancer Cell, Sturm et al. report that global DNA methylation patterns in glioblastoma multiforme divide adult and pediatric tumors into subgroups that have characteristic DNA mutations, mRNA profiles, and most importantly, different clinical behaviors. These findings suggest novel opportunities for therapeutics for this dreaded disease

Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma

Abstract Background The Sonic hedgehog (Shh) signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors. Methods We silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples. Results Silencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas. Conclusions Our results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.http://deepblue.lib.umich.edu/bitstream/2027.42/78313/1/1471-2407-10-614.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78313/2/1471-2407-10-614.pdfPeer Reviewe

Identification of astrocytoma associated genes including cell surface markers

BACKGROUND: Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE) profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide. METHODS: We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas [1]. These profiles were produced as part of the Cancer Genome Anatomy Project's SAGE Genie [2], and were used in an in silico search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors. RESULTS: A restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas (e.g. VCAM1, SMOC1, and thymidylate synthetase), with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions. CONCLUSIONS: This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes

Neoplastic cells are a rare component in human glioblastoma microvasculature

Microvascular proliferation is a key biological and diagnostic hallmark of human glioblastoma, one of the most aggressive forms of human cancer. It has recently been suggested that stem-like glioblastoma cells have the capacity to differentiate into functional endothelial cells, and that a significant proportion of the vascular lining in tumors has a neoplastic origin. In principle, this finding could significantly impact the efficacy and development of antiangiogenic therapies targeting the vasculature. While the potential of stem-like cancer cells to form endothelium in culture seems clear, in our clinical experience using a variety of molecular markers, neoplastic cells do not contribute significantly to the endothelial-lined vasculature of primary human glioblastoma. We sought to confirm this impression by analyzing vessels in glioblastoma previously examined using chromogenic in situ hybridization (CISH) for EGFR and immunohistochemistry for mutant IDH1. Vessels containing cells expressing these definitive neoplastic markers were identified in a small fraction of tumors, but only 10% of vessel profiles examined contained such cells and when identified these cells comprised less than 10% of the vascular cellularity in the cross section. Interestingly, these rare intravascular cells showing EGFR amplification by CISH or mutant IDH1 protein by immunohistochemistry were located in the middle or outer portions of vessel walls, but not amongst the morphologic boundaries of the endothelial lining. To more directly address the capacity of glioblastoma cells to contribute to the vascular endothelium, we performed double labeling (Immunofluorescence/FISH) for the endothelial marker CD34 and EGFR gene locus. Although rare CD34 positive neoplastic cells unassociated with vessels were identified (<1%), this analysis did not identify EGFR amplified cells within vascular linings, and further supports our observations that incorporation of glioblastoma cells into the tumor vessels is at best extremely rare, and therefore of questionable clinical or therapeutic significance

Isolated endogenous Nocardia endophthalmitis after immunosuppression.

PURPOSE: This study is aimed to report a case of endogenous Nocardia endophthalmitis in the setting of immunosuppression from chronic steroid use. METHODS: A case report was conducted. RESULTS: A 79-year-old woman presented with decreased vision with floaters in the left eye. Ophthalmic examination revealed severe inflammation in the anterior chamber, vitreous opacities, and retinal detachment. Vitreous cultures grew Nocardia farcinica without any systemic foci of infection found during further workup. The patient was treated with intravitreal amikacin and oral trimethoprim-sulfamethoxazole, and her retinal detachment was later repaired in the operating room. The patient has since remained stable with no signs of retinal detachment or active infection. CONCLUSIONS: Nocardia endophthalmitis is a rare, but serious intraocular infection that should be considered in the differential diagnosis in any immunosuppressed patient, including those receiving steroids, who presents with signs of intraocular infection

Isolated endogenous Nocardia endophthalmitis after immunosuppression.

PURPOSE: This study is aimed to report a case of endogenous Nocardia endophthalmitis in the setting of immunosuppression from chronic steroid use. METHODS: A case report was conducted. RESULTS: A 79-year-old woman presented with decreased vision with floaters in the left eye. Ophthalmic examination revealed severe inflammation in the anterior chamber, vitreous opacities, and retinal detachment. Vitreous cultures grew Nocardia farcinica without any systemic foci of infection found during further workup. The patient was treated with intravitreal amikacin and oral trimethoprim-sulfamethoxazole, and her retinal detachment was later repaired in the operating room. The patient has since remained stable with no signs of retinal detachment or active infection. CONCLUSIONS: Nocardia endophthalmitis is a rare, but serious intraocular infection that should be considered in the differential diagnosis in any immunosuppressed patient, including those receiving steroids, who presents with signs of intraocular infection

Embryonal tumors with abundant neuropil and true Rosettes: A distinctive CNS primitive neuroectodermal tumor

Embryonal neoplasms of the central nervous system (CNS) generally arise in the early years of life and behave in a clinically aggressive manner, but vary somewhat in their microscopic appearance. Several groups have reported examples of an embryonal tumor with combined histologic features of ependymoblastoma and neuroblastoma, a lesion referred to as "embryonal tumor with abundant neuropil and true rosettes" (ETANTR). Herein, we present 22 new cases, and additional clinical follow-up on our 7 initially reported cases, to better define the histologic features and clinical behavior of this distinctive neoplasm. It affects infants and arises most often in cerebral cortex, the cerebellum and brainstem being less frequent sites. Unlike other embryonal tumors of the CNS, girls are more commonly affected than boys. On neuroimaging, the tumors appear as large, demarcated, solid masses featuring patchy or no contrast enhancement. Five of our cases (18%) were at least partly cystic. Distinctive microscopic features include a prominent background of mature neuropil punctuated by true rosettes formed of pseudo-stratified embryonal cells circumferentially disposed about a central lumen (true rosettes). Of the 25 cases with available follow-up, 19 patients have died, their median survival being 9 months. Performed on 2 cases, cytogenetic analysis revealed extra copies of chromosome 2 in both. We believe that the ETANTR represents a histologically distinctive form of CNS embryonal tumor. ©2009 by Lippincott Williams & Wilkins

Increased 5-hydroxymethylcytosine and decreased 5-methylcytosine are indicators of global epigenetic dysregulation in diffuse intrinsic pontine glioma

Introduction Diffuse intrinsic pontine glioma (DIPG) is a malignant pediatric brain tumor associated with dismal outcome. Recent high-throughput molecular studies have shown a high frequency of mutations in histone-encoding genes (H3F3A and HIST1B) and distinctive epigenetic alterations in these tumors. Epigenetic alterations described in DIPG include global DNA hypomethylation. In addition to the generally repressive methylcytosine DNA alteration, 5-hydroxymethylation of cytosine (5hmC) is recognized as an epigenetic mark associated with active chromatin. We hypothesized that in addition to alterations in DNA methylation, that there would be changes in 5hmC. To test this hypothesis, we performed immunohistochemical studies to compare epigenetic alterations in DIPG to extrapontine adult and pediatric glioblastoma (GBM) and normal brain. A total of 124 tumors were scored for histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 9 trimethylation (H3K9me3) and 104 for 5hmC and 5-methylcytosine (5mC). An H-score was derived by multiplying intensity (0–2) by percentage of positive tumor nuclei (0-100%). Results We identified decreased H3K27me3 in the DIPG cohort compared to pediatric GBM (p \u3c 0.01), adult GBM (p \u3c 0.0001) and normal brain (p \u3c 0.0001). H3K9me3 was not significantly different between tumor types. Global DNA methylation as measured by 5mC levels were significantly lower in DIPG compared to pediatric GBM (p \u3c 0.001), adult GBM (p \u3c 0.01), and normal brain (p \u3c 0.01). Conversely, 5hmC levels were significantly higher in DIPG compared to pediatric GBM (p \u3c 0.0001) and adult GBM (p \u3c 0.0001). Additionally, in an independent set of DIPG tumor samples, TET1 andTET3 mRNAs were found to be overexpressed relative to matched normal brain. Conclusions Our findings extend the immunohistochemical study of epigenetic alterations in archival tissue to DIPG specimens. Low H3K27me3, decreased 5mC and increased 5hmC are characteristic of DIPG in comparison with extrapontine GBM. In DIPG, the relative imbalance of 5mC compared to 5hmC may represent an opportunity for therapeutic intervention