Ionizing radiation represents the most effective therapy for glioblastoma (World Health Organization grade IV glioma), one of the most lethal human malignancies, but radiotherapy remains only palliative because of radioresistance. The mechanisms underlying tumour radioresistance have remained elusive. Here we show that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of tumour cells expressing CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, is enriched after radiation in gliomas. In both cell culture and the brains of immunocompromised mice, CD133-expressing glioma cells survive ionizing radiation in increased proportions relative to most tumour cells, which lack CD133. CD133-expressing tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activate the DNA damage checkpoint in response to radiation, and repair radiation-induced DNA damage more effectively than CD133-negative tumour cells. In addition, the radioresistance of CD133-positive glioma stem cells can be reversed with a specific inhibitor of the Chk1 and Chk2 checkpoint kinases. Our results suggest that CD133-positive tumour cells represent the cellular population that confers glioma radioresistance and could be the source of tumour recurrence after radiation. Targeting DNA damage checkpoint response in cancer stem cells may overcome this radioresistance and provide a therapeutic model for malignant brain cancers.

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high-density oligonucleotide arrays, and performed gene expression analyses using next-generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 (IDH1) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs. †To whom correspondence should be addressed. E-mail: bertvog@gmail.com (B.V.); velculescu@jhmi.edu (V.E.V.); kinzlke@jhmi.edu (K.W.K.). * These authors contributed equally to this work. NIH Public Access Author ManuscriptScience. Author manuscript; available in PMC 2010 February 11. Published in final edited form as:Science. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptMalignant gliomas are the most frequent and lethal cancers originating in the central nervous system. The most biologically aggressive subtype is glioblastoma multiforme (GBM) [World Health Organization (WHO) grade IV astrocytoma], a tumor associated with a dismal prognosis (1). The current standard of care for GBM patients-surgical resection followed by adjuvant radiation therapy and chemotherapy with the oral alkylating agent temozolomideproduces a median survival of only 15 months (2). Historically, GBMs have been categorized into two groups ("primary" and "secondary") on the basis of clinical presentation (3). Secondary GBMs are defined as cancers that have clinical, radiologic, or histopathologic evidence of malignant progression from a preexisting lower-grade tumor, whereas primary GBMs have no such history and present at diagnosis as advanced cancers (4). Clinical differences have been re ported between the two groups, with secondary GBMs occurring less frequently (~5% of GBMs) and predominantly in younger patients (median age ~45 years versus ~60 years for primary GBM) (5,6). The histopathologic findings of primary and secondary GBMs are indistinguishable, and the prognosis does not appear to be different after adjustment for age (5,6).Substantial research effort has focused on the identification of genetic alterations in GBMs that might help define subclasses of GBM patients with differing prognoses and/or response to specific therapies (7). Distinctions between the genetic lesions found in primary and secondary GBMs have been made, with TP53 mutations occurring more commonly in secondary GBMs and EGFR amplifications and ...

BACKGROUND A recent genomewide mutational analysis of glioblastomas (World Health Organization [WHO] grade IV glioma) revealed somatic mutations of the isocitrate dehydrogenase 1 gene (IDH1) in a fraction of such tumors, most frequently in tumors that were known to have evolved from lower-grade gliomas (secondary glioblastomas). METHODS We determined the sequence of the IDH1 gene and the related IDH2 gene in 445 central nervous system (CNS) tumors and 494 non-CNS tumors. The enzymatic activity of the proteins that were produced from normal and mutant IDH1 and IDH2 genes was determined in cultured glioma cells that were transfected with these genes. RESULTS We identified mutations that affected amino acid 132 of IDH1 in more than 70% of WHO grade II and III astrocytomas and oligodendrogliomas and in glioblastomas that developed from these lower-grade lesions. Tumors without mutations in IDH1 often had mutations affecting the analogous amino acid (R172) of the IDH2 gene. Tumors with IDH1 or IDH2 mutations had distinctive genetic and clinical characteristics, and patients with such tumors had a better outcome than those with wild-type IDH genes. Each of four tested IDH1 and IDH2 mutations reduced the enzymatic activity of the encoded protein. CONCLUSIONS Mutations of NADP+-dependent isocitrate dehydrogenases encoded by IDH1 and IDH2 occur in a majority of several types of malignant gliomas.

We describe the landscape of somatic genomic alterations based on multi-dimensional 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.

We conducted comprehensive integrative molecular analyses of the complete set of tumors in The Cancer Genome Atlas (TCGA), consisting of approximately 10,000 specimens and representing 33 types of cancer. We performed molecular clustering using data on chromosome-arm-level aneuploidy, DNA hypermethylation, mRNA, and miRNA expression levels and reverse-phase protein arrays, of which all, except for aneuploidy, revealed clustering primarily organized by histology, tissue type, or anatomic origin. The influence of cell type was evident in DNA-methylation-based clustering, even after excluding sites with known preexisting tissue-type-specific methylation. Integrative clustering further emphasized the dominant role of cell-of-origin patterns. Molecular similarities among histologically or anatomically related cancer types provide a basis for focused pan-cancer analyses, such as pan-gastrointestinal, pan-gynecological, pan-kidney, and pan-squamous cancers, and those related by stemness features, which in turn may inform strategies for future therapeutic development.

BACKGROUND Diffuse low-grade and intermediate-grade gliomas (which together make up the lower-grade gliomas, World Health Organization grades II and III) have highly variable clinical behavior that is not adequately predicted on the basis of histologic class. Some are indolent; others quickly progress to glioblastoma. The uncertainty is compounded by interobserver variability in histologic diagnosis. Mutations in IDH, TP53, and ATRX and codeletion of chromosome arms 1p and 19q (1p/19q codeletion) have been implicated as clinically relevant markers of lower-grade gliomas. METHODS We performed genomewide analyses of 293 lower-grade gliomas from adults, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA expression, microRNA expression, and targeted protein expression. These data were integrated and tested for correlation with clinical outcomes. RESULTS Unsupervised clustering of mutations and data from RNA, DNA-copy-number, and DNA-methylation platforms uncovered concordant classification of three robust, nonoverlapping, prognostically significant subtypes of lower-grade glioma that were captured more accurately by IDH, 1p/19q, and TP53 status than by histologic class. Patients who had lower-grade gliomas with an IDH mutation and 1p/19q codeletion had the most favorable clinical outcomes. Their gliomas harbored mutations in CIC, FUBP1, NOTCH1, and the TERT promoter. Nearly all lower-grade gliomas with IDH mutations and no 1p/19q codeletion had mutations in TP53 (94%) and ATRX inactivation (86%). The large majority of lower-grade gliomas without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary glioblastoma. CONCLUSIONS The integration of genomewide data from multiple platforms delineated three molecular classes of lower-grade gliomas that were more concordant with IDH, 1p/19q, and TP53 status than with histologic class. Lower-grade gliomas with an IDH mutation either had 1p/19q codeletion or carried a TP53 mutation. Most lower-grade gliomas without an IDH mutation were molecularly and clinically similar to glioblastoma. (Funded by the National Institutes of Health.)

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Summary Glioblastomas are lethal cancers characterized by florid angiogenesis promoted in part by glioma stem cells (GSCs). As hypoxia regulates angiogenesis, we examined hypoxic responses in GSCs. We now demonstrate that hypoxia-inducible factor HIF2α and multiple HIF-regulated genes are preferentially expressed in GSCs in comparison to nonstem tumor cells and normal neural progenitors. In tumor specimens, HIF2α co-localizes with cancer stem cell markers. Targeting HIFs in GSCs inhibits self-renewal, proliferation and survival in vitro, and attenuates tumor initiation potential of GSCs in vivo. Analysis of a molecular database reveals that HIF2A expression correlates with poor glioma patient survival. Our results demonstrate that GSCs differentially respond to hypoxia with distinct HIF induction patterns and HIF2α may represent a promising target for anti-glioblastoma therapies. Significance Recent evidence supports the presence of cancer stem cell populations that contribute to tumor progression through preferential resistance to radiation and chemotherapy, and promotion of tumor angiogenesis, invasion, and metastasis. Therefore, the elucidation of molecular regulators of cancer stem cells may translate into improved anti-neoplastic therapies. Our work demonstrates that cancer stem cells derived from glioblastomas differentially respond to hypoxia with a distinct induction of HIF2α. We find that HIFs are critical to cancer stem cell maintenance and angiogenic drive, and that expression of HIF2α is significantly associated with poor glioma patient survival. These data further suggest that anti-angiogenic therapies can be designed to target cancer stem cell specific molecules involved in neoangiogenesis, including HIF2α and its regulated factors.