The somatic genomic landscape of glioblastoma.

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

Exploration of the gene fusion landscape of glioblastoma using transcriptome sequencing and copy number data.

BACKGROUND: RNA-seq has spurred important gene fusion discoveries in a number of different cancers, including lung, prostate, breast, brain, thyroid and bladder carcinomas. Gene fusion discovery can potentially lead to the development of novel treatments that target the underlying genetic abnormalities. RESULTS: In this study, we provide comprehensive view of gene fusion landscape in 185 glioblastoma multiforme patients from two independent cohorts. Fusions occur in approximately 30-50% of GBM patient samples. In the Ivy Center cohort of 24 patients, 33% of samples harbored fusions that were validated by qPCR and Sanger sequencing. We were able to identify high-confidence gene fusions from RNA-seq data in 53% of the samples in a TCGA cohort of 161 patients. We identified 13 cases (8%) with fusions retaining a tyrosine kinase domain in the TCGA cohort and one case in the Ivy Center cohort. Ours is the first study to describe recurrent fusions involving non-coding genes. Genomic locations 7p11 and 12q14-15 harbor majority of the fusions. Fusions on 7p11 are formed in focally amplified EGFR locus whereas 12q14-15 fusions are formed by complex genomic rearrangements. All the fusions detected in this study can be further visualized and analyzed using our website: http://ivygap.swedish.org/fusions. CONCLUSIONS: Our study highlights the prevalence of gene fusions as one of the major genomic abnormalities in GBM. The majority of the fusions are private fusions, and a minority of these recur with low frequency. A small subset of patients with fusions of receptor tyrosine kinases can benefit from existing FDA approved drugs and drugs available in various clinical trials. Due to the low frequency and rarity of clinically relevant fusions, RNA-seq of GBM patient samples will be a vital tool for the identification of patient-specific fusions that can drive personalized therapy.

Genome-wide analysis of epigenetic silencing identifies BEX1 and BEX2 as candidate tumor suppressor genes in malignant glioma.

Promoter hypermethylation and histone deacetylation are common epigenetic mechanisms implicated in the transcriptional silencing of tumor suppressor genes in human cancer. We treated two immortalized glioma cell lines, T98 and U87, and 10 patient-derived primary glioma cell lines with trichostatin A (TSA), a histone deacetylase inhibitor, or 5-aza-2'-deoxycytidine (5-AzaC), a DNA methyltransferase inhibitor, to comprehensively identify the cohort of genes reactivated through the pharmacologic reversal of these distinct but related epigenetic processes. Whole-genome microarray analysis identified genes induced by TSA (653) or 5-AzaC treatment (170). We selected a subset of reactivated genes that were markedly induced (greater than two-fold) after treatment with either TSA or 5-AzaC in a majority of glioma cell lines but not in cultured normal astrocytes. We then characterized the degree of promoter methylation and transcriptional silencing of selected genes in histologically confirmed human tumor and nontumor brain specimens. We identified two novel brain expressed genes, BEX1 and BEX2, which were silenced in all tumor specimens and exhibited extensive promoter hypermethylation. Viral-mediated reexpression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis and potent tumor suppressor effects in vitro and in a xenograft mouse model. Using an integrated approach, we have established a novel platform for the genome-wide screening of epigenetically silenced genes in malignant glioma. This experimental paradigm provides a powerful new method for the identification of epigenetically silenced genes with potential function as tumor suppressors, biomarkers for disease diagnosis and detection, and therapeutically reversible modulators of critical regulatory pathways important in glioma pathogenesis.

Dickkopf-1 is an epigenetically silenced candidate tumor suppressor gene in medulloblastoma.

Medulloblastoma is a heterogeneous pediatric brain tumor with significant therapy-related morbidity, its five-year survival rates ranging from 30% to 70%. Improvement in diagnosis and therapy requires better understanding of medulloblastoma pathology. We used whole-genome microarray analysis to identify putative tumor suppressor genes silenced by epigenetic mechanisms in medulloblastoma. This analysis yielded 714 up-regulated genes in immortalized medulloblastoma cell line D283 on treatment with histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Dickkopf-1 (DKK1), a Wnt antagonist, was found to be up-regulated on HDAC inhibition. We examined DKK1 expression in primary medulloblastoma cells and patient samples by reverse transcriptase PCR and found it to be significantly down-regulated relative to normal cerebellum. Transfection of a DKK1 gene construct into D283 cell lines suppressed medulloblastoma tumor growth in colony focus assays by 60% (P < 0.001). In addition, adenoviral vector-mediated expression of DKK1 in medulloblastoma cells increased apoptosis fourfold (P < 0.001). These data reveal that inappropriate histone modifications might deregulate DKK1 expression in medulloblastoma tumorigenesis and block its tumor-suppressive activity.

A Cell-Surface Membrane Protein Signature for Glioblastoma.

We present a systems strategy that facilitated the development of a molecular signature for glioblastoma (GBM), composed of 33 cell-surface transmembrane proteins. This molecular signature, GBMSig, was developed through the integration of cell-surface proteomics and transcriptomics from patient tumors in the REMBRANDT (n = 228) and TCGA datasets (n = 547) and can separate GBM patients from control individuals with a Matthew's correlation coefficient value of 0.87 in a lock-down test. Functionally, 17/33 GBMSig proteins are associated with transforming growth factor ß signaling pathways, including CD47, SLC16A1, HMOX1, and MRC2. Knockdown of these genes impaired GBM invasion, reflecting their role in disease-perturbed changes in GBM. ELISA assays for a subset of GBMSig (CD44, VCAM1, HMOX1, and BIGH3) on 84 plasma specimens from multiple clinical sites revealed a high degree of separation of GBM patients from healthy control individuals (area under the curve is 0.98 in receiver operating characteristic). In addition, a classifier based on these four proteins differentiated the blood of pre- and post-tumor resections, demonstrating potential clinical value as biomarkers.

Genetic investigation of multicentric glioblastoma multiforme: case report.

The authors report a case of multicentric glioblastoma multiforme (GBM) in which all 4 tumor foci were resected and evaluated using both comparative genomic hybridization array and RNA sequencing. Genetic analysis showed that the tumors shared a common origin, although each had its own unique set of genetic aberrations. The authors note that the genetic heterogeneity of multicentric GBM likely contributes to the failures of current treatments. The case underscores the necessity of increased genetic investigation.

Cellular pleomorphism in papillary tumors of the pineal region.

Papillary tumor of the pineal region (PTPR) is a recently recognized entity. We present the pathologic findings of two cases of PTPR as examples, and discuss the presence of cellular pleomorphism in these tumors. Patient 1 is a 48-year-old man with a pineal region mass. The tumor had unique biphasic patterns, papillary/pseudopapillary areas, and increased mitotic activity. Juxtaposed areas had marked pleomorphism, including nuclear enlargement, smudgy chromatin, nuclear pseudoinclusions, and cytoplasmic vacuolation. Mitoses were absent in these areas. Immunohistochemical staining revealed strong S100 expression. CAM 5.2 and CK18 were strongly positive in a patchy fashion. MIB1 labeling indices were high in classic PTPR regions but very low in pleomorphic areas. Patient 2 was a 35-year-old male with a pineal region tumor characterized by papillary architecture and overall cellular monotony, rare mitoses, and pleomorphism as a more isolated finding, with associated nuclear enlargement and crowding. S100 and CAM 5.2 labeling were present, and MIB1 labeling index was very low throughout the tumor. We discuss the pathologic and phenotypic features of PTPR. Variable pleomorphism may be present, reflected in size variation and nuclear hyperchromasia, but was not accompanied by increased proliferative activity in these cases, suggesting a degenerative phenomenon.

High-throughput chemical screens identify disulfiram as an inhibitor of human glioblastoma stem cells.

Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind, we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation, of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin, deguelin, patulin and phenethyl caffeate) exhibited high cytotoxicity, with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular, the FDA approved drug for the treatment of alcoholism, disulfiram (DSF), was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu), which markedly increased GSC death. DSF-Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs, consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier, we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM.

Comprehensive analysis of MGMT promoter methylation: correlation with MGMT expression and clinical response in GBM.

O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089-13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301-7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting.

Epigenetic regulation of wnt pathway antagonists in human glioblastoma multiforme.

Epigenetic inactivation of tumor suppressor genes is common in human cancer. Using a large-scale whole-genome approach in an earlier study, the authors identified epigenetically silenced genes with potential tumor suppressor function in glioblastoma (GBM). Three genes identified in this analysis-DKK1, SFRP1, and WIF1-are potent inhibitors of the Wnt signal transduction pathway. Here, the authors confirm decreased expression of these genes in GBM tumor tissue samples relative to nontumor brain tissue samples using real-time PCR. They then show that expression of all 3 genes is restored in T98 GBM cells by treatment with the histone deacetylase inhibitor Trichostatin A (TSA), but only DKK1 expression is restored by treatment with the demethylating agent 5-azacytidine. Bisulfite sequencing did not reveal significant methylation in the promoter region of DKK1, whereas histone acetylation and chromatin accessibility increased significantly for all 3 genes after TSA treatment. Ectopic expression of DKK1 significantly reduces colony formation and increases chemotherapy-induced apoptosis in T98 cells. Ectopic expression of the canonical Wnt pathway inhibitors WIF1 and SFRP1 shows a relative lack of response. Chronic Wnt3a stimulation only partially reverses growth suppression after DKK1 reexpression, whereas a specific inhibitor of the JNK pathway significantly reverses the effect of DKK1 reexpression on colony formation and apoptosis in T98 cells. These results support a potential growth-suppressive function for epigenetically silenced DKK1 in GBM and suggest that DKK1 restoration could modulate Wnt signaling through both canonical and noncanonical pathways.

Bioprocessing of human glioblastoma brain cancer tissue.

Solid cancer tumors are thought to arise from aberrant stem cell populations, called cancer stem cells (CSCs). Hence, the development of effective cancer therapies may rely on developing methods that specifically target these cells. However, the scarcity of CSCs in vivo represents a major impediment to such research, as there is an insufficient supply for basic biochemical and genetic analyses. It is therefore necessary to develop methods to expand reproducibly CSC tissue in vitro in a controlled environment. To date, we have developed bioreactor protocols for the suspension culture of an aggressive and deadly type of brain cancer called glioblastoma multiforme (GBM). Human GBM-derived cells achieved a maximum cell density of 2.4 x 10(6) cells/mL after 24 days under high shear conditions in batch culture conditions. In comparison, fed-batch cultures achieved 4.5 x 10(6) cells/mL after 32 days. Characterization of bioreactor-expanded cells using both flow cytometry and a differentiation assay indicated that bioreactor-generated human GBM-derived cells have similar characteristics to the initial cell population and achieve >90% CD133 expression. Additionally, genomic characterization indicated that a very small number of key genes were differentially expressed in the bioreactor-expanded GBM-derived cells, thereby conserving the basic nature of the brain cancer tissue in the cell expansion process.

Targeting stem cells-clinical implications for cancer therapy.

Cancer stem cells (CSC), also called tumor initiating cells (TIC), are considered to be the origin of replicating malignant tumor cells in a variety of human cancers. Their presence in the tumor may herald malignancy potential, mediate resistance to conventional chemotherapy or radiotherapy, and confer poor survival outcomes. Thus, CSC may serve as critical cellular targets for treatment. The ability to therapeutically target CSC hinges upon identifying their unique cell surface markers and the underlying survival signaling pathways. While accumulating evidence suggests cell-surface antigens (such as CD44, CD133) as CSC markers for several tumor tissues, emerging clinical needs exist for the identification of new markers to completely separate CSC from normal stem cells. Recent studies have demonstrated the critical role of the tumor suppressor PTEN/PI3 kinase pathway in regulating TIC in leukemia, brain, and intestinal tissues. The successful eradication of tumors by therapies targeting CSC will require an in-depth understanding of the molecular mechanisms governing CSC self renewal, differentiation, and escape from conventional therapy. Here we review recent progress from brain tumor and intestinal stem cell research with a focus on the PTEN-Akt-Wnt pathway, and how the components of CSC pathways may serve as biomarkers for diagnosis, prognosis, and therapeutics.

Systems biology and cancer stem cells.

The identification, purification, and characterization of cancer stem cells holds tremendous promise for improving the treatment of cancer. Mounting evidence is demonstrating that only certain tumor cells (i.e. the cancer stem cells) can give rise to tumors when injected and that these purified cell populations generate heterogeneous tumors. While the cell of origin is still not determined definitively, specific molecular markers for populations containing these cancer stem cells have been found for leukemia, brain cancer, and breast cancer, among others. Systems approaches, particularly molecular profiling, have proven to be of great utility for cancer diagnosis and characterization. These approaches also hold significant promise for identifying distinctive properties of the cancer stem cells, and progress is already being made.