Identification of driver mutations in human diseases is often limited by cohort size and availability of appropriate statistical models. We propose a framework for the systematic discovery of genetic ...alterations that are causal determinants of disease, by prioritizing genes upstream of functional disease drivers, within regulatory networks inferred de novo from experimental data. We tested this framework by identifying the genetic determinants of the mesenchymal subtype of glioblastoma. Our analysis uncovered KLHL9 deletions as upstream activators of two previously established master regulators of the subtype, C/EBPβ and C/EBPδ. Rescue of KLHL9 expression induced proteasomal degradation of C/EBP proteins, abrogated the mesenchymal signature, and reduced tumor viability in vitro and in vivo. Deletions of KLHL9 were confirmed in > 50% of mesenchymal cases in an independent cohort, thus representing the most frequent genetic determinant of the subtype. The method generalized to study other human diseases, including breast cancer and Alzheimer’s disease.
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•Combining cellular networks, gene expression, and genomic data finds new mutations•KLHL9 deletions predict mesenchymal transformation and poorest prognosis in GBM•KLHL9 posttranslationally regulates CEBPβ/δ, and rescue inhibits tumor growth•DIGGIT can be used on any genetic disease with matched expression and genomic data
An algorithm that systematically analyzes regulatory and signaling networks upstream of master regulator genes by combining cellular networks, gene expression, and genomic data allows the identification of genetic alterations driving complex diseases such as cancer and Alzheimer’s disease.
Recent molecular classification of glioblastoma (GBM) has shown that patients with a mesenchymal (MES) gene expression signature exhibit poor overall survival and treatment resistance. Using ...regulatory network analysis of available expression microarray data sets of GBM, including The Cancer Genome Atlas (TCGA), we identified the transcriptional coactivator with PDZ-binding motif (TAZ), to be highly associated with the MES network. TAZ expression was lower in proneural (PN) GBMs and lower-grade gliomas, which correlated with CpG island hypermethylation of the TAZ promoter compared with MES GBMs. Silencing of TAZ in MES glioma stem cells (GSCs) decreased expression of MES markers, invasion, self-renewal, and tumor formation. Conversely, overexpression of TAZ in PN GSCs as well as murine neural stem cells (NSCs) induced MES marker expression and aberrant osteoblastic and chondrocytic differentiation in a TEAD-dependent fashion. Using chromatin immunoprecipitation (ChIP), we show that TAZ is directly recruited to a majority of MES gene promoters in a complex with TEAD2. The coexpression of TAZ, but not a mutated form of TAZ that lacks TEAD binding, with platelet-derived growth factor-B (PDGF-B) resulted in high-grade tumors with MES features in a murine model of glioma. Our studies uncover a direct role for TAZ and TEAD in driving the MES differentiation of malignant glioma.
Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating ...agent temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O⁶-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N⁷-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.
Isocitrate dehydrogenase (IDH) mutational testing is becoming increasingly important. For this, robust and reliable assays are needed. We tested the variation of results between six laboratories of ...testing for IDH mutations. Each laboratory received five unstained slides from 31 formalin-fixed paraffin-embedded (FFPE) glioma samples, and followed its own standard IDH diagnostic routine. All laboratories used immunohistochemistry (IHC) with an antibody against the most frequent IDH1 mutation (R132H) as a first step. Three laboratories then sequenced only IHC negative cases while the others sequenced all cases. Based on the overall analysis, 13 samples from 11 tumors had an R132H mutation and one tumor showed an R132G mutation. Results of IHC for IDH1 R132H mutations in all six laboratories were completely in agreement, and identified all R132H mutations. Upon sequencing the results of two laboratories deviated from those of the others. After a review of the entire diagnostic process, on repeat (blinded) testing one laboratory was completely in agreement with the overall result. A change in technique did only partially improve the results in the other laboratory. IHC for the IDH1 R132H mutation is very reliable and consistent across laboratories. IDH sequencing procedures yielded inconsistent results in 2 out of 6 laboratories. Quality assurance is pivotal before IDH testing is made part of clinical management of patients.
Diffuse gliomas are up till now graded based upon morphology. Recent findings indicate that isocitrate dehydrogenase (IDH) mutation status defines biologically distinct groups of tumors. The role of ...tumor grade and mitotic index in patient outcome has not been evaluated following stratification by IDH mutation status. To address this, we interrogated 558 WHO grade II–III diffuse gliomas for
IDH1/2
mutations and investigated the prognostic impact of WHO grade within IDH-mutant and IDH-wild type tumor subsets independently. The prognostic impact of grade was modest in IDH-mutant hazard ratio (HR) = 1.21, 95 % confidence interval (CI) = 0.91–1.61 compared to IDH-wild type tumors (HR = 1.74, 95 % CI = 0.95–3.16). Using a dichotomized mitotic index cut-off of 4/1000 tumor cells, we found that while mitotic index was significantly associated with outcome in IDH-wild type tumors (log-rank
p
< 0.0001, HR = 4.41, 95 % CI = 2.55–7.63), it was not associated with outcome in IDH-mutant tumors (log-rank
p
= 0.5157, HR = 1.10, 95 % CI = 0.80–1.51), and could demonstrate a statistical interaction (
p
< 0.0001) between IDH mutation and mitotic index (i.e., suggesting that the effect of mitotic index on patient outcome is dependent on IDH mutation status). Patient age, an established prognostic factor in diffuse glioma, was significantly associated with outcome only in the IDH-wild type subset, and consistent with prior data, 1p/19q co-deletion conferred improved outcome in the IDH-mutant cohort. These findings suggest that stratification of grade II–III gliomas into subsets defined by the presence or absence of IDH mutation leads to subgroups with distinct prognostic characteristics. Further evaluation of grading criteria and prognostic markers is warranted within IDH-mutant versus IDH-wild type diffuse grade II–III gliomas as independent entities.
We have profiled promoter DNA methylation alterations in 272 glioblastoma tumors in the context of The Cancer Genome Atlas (TCGA). We found that a distinct subset of samples displays concerted ...hypermethylation at a large number of loci, indicating the existence of a glioma-CpG island methylator phenotype (G-CIMP). We validated G-CIMP in a set of non-TCGA glioblastomas and low-grade gliomas. G-CIMP tumors belong to the proneural subgroup, are more prevalent among lower-grade gliomas, display distinct copy-number alterations, and are tightly associated with
IDH1 somatic mutations. Patients with G-CIMP tumors are younger at the time of diagnosis and experience significantly improved outcome. These findings identify G-CIMP as a distinct subset of human gliomas on molecular and clinical grounds.
► Identification of a CpG island methylator phenotype (G-CIMP) in gliomas ► G-CIMP is tightly associated with
IDH1 mutation ► G-CIMP patients are younger at diagnosis and display improved survival ► G-CIMP is more prevalent among low- and intermediate-grade gliomas
The histopathological diagnosis of diffuse gliomas often lacks the precision that is needed for tailored treatment of individual patients. Assessment of the molecular aberrations will probably allow ...more robust and prognostically relevant classification of these tumors. Markers that have gained a lot of interest in this respect are co-deletion of complete chromosome arms 1p and 19q, (hyper)methylation of the MGMT promoter and IDH1 mutations. The aim of this study was to assess the prognostic significance of complete 1p/19q co-deletion, MGMT promoter methylation and IDH1 mutations in patients suffering from diffuse gliomas. The presence of these molecular aberrations was investigated in a series of 561 diffuse astrocytic and oligodendroglial tumors (low grade n=110, anaplastic n=118 and glioblastoma n=333) and correlated with age at diagnosis and overall survival. Complete 1p/19q co-deletion, MGMT promoter methylation and/or IDH1 mutation generally signified a better prognosis for patients with a diffuse glioma including glioblastoma. However, in all 10 patients with a histopathological diagnosis of glioblastoma included in this study complete 1p/19q co-deletion was not associated with improved survival. Furthermore, in glioblastoma patients >50 years of age the favorable prognostic significance of IDH1 mutation and MGMT promoter methylation was absent. In conclusion, molecular diagnostics is a powerful tool to obtain prognostically relevant information for glioma patients. However, for individual patients the molecular information should be interpreted with caution and weighed in the context of parameters such as age and histopathological diagnosis.
BACKGROUND: Both IDH mutated (IDHmut) and wild-type (IDHwt) lower grade gliomas can progress to GBM. However, a detailed study of alterations associated with progression of these molecularly distinct ...tumor types has not been described. Here we perform an analysis of copy number alterations (CNA) across all grades (Grade II-II and Grade IV) IDHmut vs IDHwt infiltrating gliomas. METHODS: DNA was extracted from 94 patient FFPE glioma samples from 4 clinical and molecular groups: Grade II-III IDHwt (n = 17), Grade II-III IDHmut (n = 28), Grade IV IDHwt (n = 25), and Grade IV IDHmut (n = 24). CNA were detected by molecular inversion probes (OncoScan FFPE Express, Affymetrix) and analyzed with Nexus Copy Number Software (BioDiscovery). GISTIC was used to define deletions and amplifications. Chromothripsis ("chromosomal shattering") was defined using stringent criteria of at least ten switches of CNA in individual chromosomes. RESULTS: Unsupervised clustering of CNAs demonstrated distinct clusters within IDHmut gliomas that correlated with grade. However, within IDHwt gliomas all grades clustered together regardless of grade, with Chr7 amplification (including EGFR) and loss of Chr10 (including PTEN) seen in most tumors. IDHwt Grade II-III and Grade IV tumors both displayed relatively poor prognosis (median survivals of 65.4 and 37.4 weeks). However, IDHmut gliomas had better survival for all grades (604.3 weeks for Grade II-III and 270.3 weeks for Grade IV). Grade IV IDHmut gliomas were more likely to have gains of 1q25.3 (SMG7, NCF2), 1q32.1 (KIF14, DDX59, BTG2), 6p21.1 (HSP90AB1 and other genes) and loss of 3p21 compared with Grade II-III. Functional analyses showed that IDHwt tumors had more amplifications in receptor tyrosine kinases and their downstream pathways. In terms of novel prognostic markers within IDHmut Grade II-III tumors, multivariate analysis identified loss of estrogen receptor B and loss of 10q26.3 containing part of GLRX3 as poor prognostic factors, and CDKN1C loss as a good prognostic factor. Finally, significantly higher incidence of chromothripsis events were observed in grade IV IDHmut compared to IDHwt. CONCLUSIONS: CNA analysis demonstrated significant differences in molecular ontogeny, progression, and prognosis between IDHwt and IDHmut gliomas, which may serve to further elucidate pathogenesis of these distinct tumor types. Significant CNA increases and increased chromothripsis in grade IV IDHmut support malignant transformation of IDHmut low grade gliomas through accumulation of genomic instability, that results later in partial overlap of CNA alterations that are seen earlier in the development of IDHwt tumors. SECONDARY CATEGORY: Tumor Biology.
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