Deep sequencing of a recurrent oligodendroglioma and the derived xenografts reveals new insights into the evolution of human oligodendroglioma and candidate driver genes.

We previously reported the establishment of a rare xenograft derived from a recurrent oligodendroglioma with 1p/19q codeletion. Here, we analyzed in detail the exome sequencing datasets from the recurrent oligodendroglioma (WHO grade III, recurrent O2010) and the first-generation xenograft (xenograft1). Somatic SNVs and small InDels (n = 80) with potential effects at the protein level in recurrent O2010 included variants in IDH1 (NM_005896:c.395G>A; p. Arg132His), FUBP1 (NM_003902:c.1307_1310delTAGA; p.Ile436fs), and CIC (NM_015125:c.4421T>G; p.Val1474Gly). All but 2 of these 80 variants were also present in xenograft1, along with 7 new variants. Deep sequencing of the 87 SNVs and InDels in the original tumor (WHO grade III, primary O2005) and in a second-generation xenograft (xenograft2) revealed that only 11 variants, including IDH1 (NM_005896:c.395G>A; p. Arg132His), PSKH1 (NM_006742.2:c.650G>A; p.Arg217Gln), and SNX12 (NM_001256188:c.470G>A; p.Arg157His), along with a variant in the TERT promoter (C250T, NM_198253.2: c.-146G>A), were already present in primary O2005. Allele frequencies of the 11 variants were calculated to assess their potential as putative driver genes. A missense change in NDST4 (NM_022569:c.2392C>G; p.Leu798Val) on 4q exhibited an increasing allele frequency (~ 20%, primary O2005, 80%, recurrent O2010 and 100%, xenograft1), consistent with a selection event. Sequencing of NDST4 in a cohort of 15 oligodendrogliomas, however, revealed no additional cases with potential protein disrupting variants. Our analysis illuminated a tumor evolutionary series of events, which included 1p/19q codeletion, IDH1 R132H, and TERT C250T as early events, followed by loss of function of NDST4 and mutations in FUBP1 and CIC as late events.

Tumor versus stromal cells in culture--survival of the fittest?

Two of the signature genetic events that occur in human gliomas, EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.

Novel CIC point mutations and an exon-spanning, homozygous deletion identified in oligodendroglial tumors by a comprehensive genomic approach including transcriptome sequencing.

Oligodendroglial tumors form a distinct subgroup of gliomas, characterized by a better response to treatment and prolonged overall survival. Most oligodendrogliomas and also some oligoastrocytomas are characterized by a unique and typical unbalanced translocation, der(1,19), resulting in a 1p/19q co-deletion. Candidate tumor suppressor genes targeted by these losses, CIC on 19q13.2 and FUBP1 on 1p31.1, were only recently discovered. We analyzed 17 oligodendrogliomas and oligoastrocytomas by applying a comprehensive approach consisting of RNA expression analysis, DNA sequencing of CIC, FUBP1, IDH1/2, and array CGH. We confirmed three different genetic subtypes in our samples: i) the "oligodendroglial" subtype with 1p/19q co-deletion in twelve out of 17 tumors; ii) the "astrocytic" subtype in three tumors; iii) the "other" subtype in two tumors. All twelve tumors with the 1p/19q co-deletion carried the most common IDH1 R132H mutation. In seven of these tumors, we found protein-disrupting point mutations in the remaining allele of CIC, four of which are novel. One of these tumors also had a deleterious mutation in FUBP1. Only by integrating RNA expression and array CGH data, were we able to discover an exon-spanning homozygous microdeletion within the remaining allele of CIC in an additional tumor with 1p/19q co-deletion. Therefore we propose that the mutation rate might be underestimated when looking at sequence variants alone. In conclusion, the high frequency and the spectrum of CIC mutations in our 1p/19q-codeleted tumor cohort support the hypothesis that CIC acts as a tumor suppressor in these tumors, whereas FUBP1 might play only a minor role.

A novel, diffusely infiltrative xenograft model of human anaplastic oligodendroglioma with mutations in FUBP1, CIC, and IDH1.

Oligodendroglioma poses a biological conundrum for malignant adult human gliomas: it is a tumor type that is universally incurable for patients, and yet, only a few of the human tumors have been established as cell populations in vitro or as intracranial xenografts in vivo. Their survival, thus, may emerge only within a specific environmental context. To determine the fate of human oligodendroglioma in an experimental model, we studied the development of an anaplastic tumor after intracranial implantation into enhanced green fluorescent protein (eGFP) positive NOD/SCID mice. Remarkably after nearly nine months, the tumor not only engrafted, but it also retained classic histological and genetic features of human oligodendroglioma, in particular cells with a clear cytoplasm, showing an infiltrative growth pattern, and harboring mutations of IDH1 (R132H) and of the tumor suppressor genes, FUBP1 and CIC. The xenografts were highly invasive, exhibiting a distinct migration and growth pattern around neurons, especially in the hippocampus, and following white matter tracts of the corpus callosum with tumor cells accumulating around established vasculature. Although tumors exhibited a high growth fraction in vivo, neither cells from the original patient tumor nor the xenograft exhibited significant growth in vitro over a six-month period. This glioma xenograft is the first to display a pure oligodendroglioma histology and expression of R132H. The unexpected property, that the cells fail to grow in vitro even after passage through the mouse, allows us to uniquely investigate the relationship of this oligodendroglioma with the in vivo microenvironment.

EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis.

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.

In vivo models of primary brain tumors: pitfalls and perspectives.

Animal modeling for primary brain tumors has undergone constant development over the last 60 years, and significant improvements have been made recently with the establishment of highly invasive glioblastoma models. In this review we discuss the advantages and pitfalls of model development, focusing on chemically induced models, various xenogeneic grafts of human cell lines, including stem cell-like cell lines and biopsy spheroids. We then discuss the development of numerous genetically engineered models available to study mechanisms of tumor initiation and progression. At present it is clear that none of the current animal models fully reflects human gliomas. Yet, the various model systems have provided important insight into specific mechanisms of tumor development. In particular, it is anticipated that a combined comprehensive knowledge of the various models currently available will provide important new knowledge on target identification and the validation and development of new therapeutic strategies.

Identification of IGF2 signaling through phosphoinositide-3-kinase regulatory subunit 3 as a growth-promoting axis in glioblastoma.

Amplification or overexpression of growth factor receptors is a frequent occurrence in malignant gliomas. Using both expression profiling and in situ hybridization, we identified insulin-like growth factor 2 (IGF2) as a marker for a subset of glioblastomas (GBMs) that lack amplification or overexpression of EGF receptor. Among 165 primary high-grade astrocytomas, 13% of grade IV tumors and 2% of grade III tumors expressed IGF2 mRNA levels >50-fold the sample population median. IGF2-overexpressing tumors frequently displayed PTEN loss, were highly proliferative, exhibited strong staining for phospho-Akt, and belonged to a subclass of GBMs characterized by poor survival. Using a serum-free culture system, we discovered that IGF2 can substitute for EGF to support the growth of GBM-derived neurospheres. The growth-promoting effects of IGF2 were mediated by the insulin-like growth factor receptor 1 and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), a regulatory subunit of phosphoinositide 3-kinase that shows genomic gains in some highly proliferative GBM cases. PIK3R3 knockdown inhibited IGF2-induced growth of GBM-derived neurospheres. The current results provide evidence that the IGF2-PIK3R3 signaling axis is involved in promoting the growth of a subclass of highly aggressive human GBMs that lack EGF receptor amplification. Our data underscore the importance of the phosphoinositide 3-kinase/Akt pathway for growth of high-grade gliomas and suggest that multiple molecular alterations that activate this signaling cascade may promote tumorigenesis. Further, these findings highlight the parallels between growth factors or receptors that are overexpressed in GBMs and those that support in vitro growth of tumor-derived stem-like cells.

Contribution of Notch signaling activation to human glioblastoma multiforme.

OBJECT: Because activation of Notch receptors has been suggested to be critical for Ras-mediated transformation, and because many gliomas exhibit deregulated Ras signaling, the authors measured Notch levels and activation in primary samples and cell lines derived from glioblastoma multiforme (GBM) as well as the contribution of Notch pathway activation to astrocytic transformation and growth. METHODS: Western blot analysis of Notch 1 expression and activation showed that Notch 1 protein was overexpressed and/or activated in Ras-transformed astrocytes, in three of four GBM cell lines, and in four of five primary GBM samples. Expansion of these studies to assess mRNA expression of components of the Notch signaling pathway by cDNA expression array showed that cDNAs encoding components of the Notch signaling pathway, including the Notch ligand Jagged-1, Notch 3, and the downstream targets of Notch (HES1 and HES2), were also overexpressed relative to non-neoplastic brain controls in 23, 71, and 51% of 35 primary GBMs, respectively. Furthermore, inhibition of Notch signaling by genetic or pharmacological means led to selective suppression of the growth and expression of markers of differentiation in cells exhibiting Notch pathway deregulation. CONCLUSIONS: Notch activation contributes to Ras-induced transformation of glial cells and to glioma growth, survival, or both and as such may represent a new target for GBM therapy.

Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis.

Previously undescribed prognostic subclasses of high-grade astrocytoma are identified and discovered to resemble stages in neurogenesis. One tumor class displaying neuronal lineage markers shows longer survival, while two tumor classes enriched for neural stem cell markers display equally short survival. Poor prognosis subclasses exhibit markers either of proliferation or of angiogenesis and mesenchyme. Upon recurrence, tumors frequently shift toward the mesenchymal subclass. Chromosomal locations of genes distinguishing tumor subclass parallel DNA copy number differences between subclasses. Functional relevance of tumor subtype molecular signatures is suggested by the ability of cell line signatures to predict neurosphere growth. A robust two-gene prognostic model utilizing PTEN and DLL3 expression suggests that Akt and Notch signaling are hallmarks of poor prognosis versus better prognosis gliomas, respectively.

Integrated array-comparative genomic hybridization and expression array profiles identify clinically relevant molecular subtypes of glioblastoma.

Glioblastoma, the most aggressive primary brain tumor in humans, exhibits a large degree of molecular heterogeneity. Understanding the molecular pathology of a tumor and its linkage to behavior is an important foundation for developing and evaluating approaches to clinical management. Here we integrate array-comparative genomic hybridization and array-based gene expression profiles to identify relationships between DNA copy number aberrations, gene expression alterations, and survival in 34 patients with glioblastoma. Unsupervised clustering on either profile resulted in similar groups of patients, and groups defined by either method were associated with survival. The high concordance between these separate molecular classifications suggested a strong association between alterations on the DNA and RNA levels. We therefore investigated relationships between DNA copy number and gene expression changes. Loss of chromosome 10, a predominant genetic change, was associated not only with changes in the expression of genes located on chromosome 10 but also with genome-wide differences in gene expression. We found that CHI3L1/YKL-40 was significantly associated with both chromosome 10 copy number loss and poorer survival. Immortalized human astrocytes stably transfected with CHI3L1/YKL-40 exhibited changes in gene expression similar to patterns observed in human tumors and conferred radioresistance and increased invasion in vitro. Taken together, the results indicate that integrating DNA and mRNA-based tumor profiles offers the potential for a clinically relevant classification more robust than either method alone and provides a basis for identifying genes important in glioma pathogenesis.