Molecular Subtypes Of Glioma Research Articles

Abstract LB-004: Molecular hallmarks of cancer: Stemness

Abstract Stemness, defined as the potential for self-renewal and de-differentiation from the cell-of-origin, has been initially attributed to normal stem cells that possess the ability to give rise to all cell types in the adult organism. Cancer stem cells have been identified in most if not all hematological malignancies as well as in solid tumors. CSCs are postulated to be amongst the most resistant cells to various forms of chemotherapy and radiotherapy and contribute to relapse and metastasis and hence inferior clinical outcomes. Here, we have performed a comprehensive and multi-platform analyses of the stemness features in 33 cancer types totalling more than 10,000 mostly primary, but also some metastatic and recurrent samples. In the first step, we have derived signatures to measure the stemness using molecular profiles of normal cells with various degrees of stemness from publicly available datasets. By multiplatform analyses of the transcriptome, methylome, and chromatin markers using different machine learning computational approaches, we have obtained four independent stemness scores. Initial validation revealed comparable performance of these computational metrics for sorting the TCGA samples. The cancer types with previously documented features of de-differentiation have higher stemness score in contrast to more differentiated cancer types. More detailed analyses of recently published molecular subtypes of gliomas have revealed a strong association of high stemness scores with the worst clinical outcome, further confirming our initial hypotheses. By adopting machine learning algorithms, we have counted percentage of immune cells of both innate and acquired response infiltrating tumor tissue. Further deconvolution of the TCGA cancers allowed quantification of the tumour microenvironment composed of cancer-associated fibroblasts and the endothelial cells. Integration of gene expression and DNA methylation datasets defines classic hallmarks of pluripotent signatures which shed new light into biological processes regulating and maintaining oncogenic dedifferentiation. Our ongoing analyses of the TCGA samples sorted by the obtained metrics involve: (1) activity of the selected hallmarks of cancer that have been shown as features of the CSCs; (2) correlation with the tumor pathology grading and clinical outcomes; (3) identification of potential drivers for development and/or repositioning of drugs targeting tumor self-renewal and de-differentiation potential; and (4) development of novel biomarkers for selection of patients that will respond to these novel therapies. In summary, we defined molecular signatures of cancer stem cells that enabled classification of TCGA cancer types and identification of tumors with stem/progenitor-like phenotypes, associated with poor clinical outcomes. Understanding stemness-like hallmark of cancer will pave the way for novel diagnostics and therapies for cancer patients. Citation Format: Tathiane Malta, Artem Sokolov, Andrew J. Gentles, Tomasz Burzykowski, Olivier Gevaert, The Stemness Analysis Working Group TCGA PanCancerNetwork, Peter W. Laird, Houtan Noushmehr, Maciej Wiznerowicz. Molecular hallmarks of cancer: Stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-004. doi:10.1158/1538-7445.AM2017-LB-004

GENT-34. EPIGENOMIC STEMNESS SIGNATURE ASSOCIATED WITH GLIOMA MOLECULAR SUBTYPES

Despite the past significant progress, glioma field faces suboptimal disease classification which impact patient management and treatment. Recent study about genomic and epigenomic landscape of gliomas showed a distinct subtype of IDH-mutant, named G-CIMP-low, with loss of DNA methylation at SOX-family binding sites in intergenic regions, and overexpression of cell cycle genes, suggesting a stem cell phenotype. This subgroup was associated with poor survival. Alongside, a stem cell-like phenotype in various cancer is directly correlated with increased aggressiveness. We hypothesize that a stem cell signature can identify subtypes of glioma associated with poor outcome and improve our comprehension of glioma biology and classification. We analysed molecular profiles from 686 TCGA gliomas (532 LGG; 154 GBM) in order to score the features of tissue de-differentiation. We derived metrics to measure undifferentiated states in tumor samples to identify glioma samples with cancer stem cell phenotype. We observed that gliomas with higher de-differentiation scores were associated with higher tumor purity (p<0.001) and grade (p<0.001). Interestingly, by sorting tumors by their cancer stem cell phenotype we observed a strrong association with the recently described glioma molecular subtypes (p<0.001), which in turn is associated with the clinical outcomes (PFS, OS). The G-CIMP-low, Classical-like, Mesenchymal-like and LGm6-GBM samples, which have the worse outcomes, presented the highest cancer stem cell score. We defined molecular signatures of cancer stem cells that enabled classification of gliomas and identification of tumors with stem/progenitor-like phenotypes, associated with poor clinical outcome. Understanding undifferentiated phenotypes of glioma cells can improve our understanding of tumor biology and my pave the way for novel diagnostics and therapies for cancer patients.

A three ion channel genes-based signature predicts prognosis of primary glioblastoma patients and reveals a chemotherapy sensitive subtype.

Increasing evidence suggests that ion channels not only regulate electric signaling in excitable cells but also play important roles in the development of brain tumor. However, the roles of ion channels in glioma remain controversial. In the present study, we systematically analyzed the expression patterns of ion channel genes in a cohort of Chinese patients with glioma using RNAseq expression profiling. First, a molecular signature comprising three ion channel genes (KCNN4, KCNB1 and KCNJ10) was identified using Univariate Cox regression and two-tailed student's t test conducted in overall survival (OS) and gene expression. We assigned a risk score based on three ion channel genes to each primary Glioblastoma multiforme (pGBM) patient. We demonstrated that pGBM patients who had a high risk of unfavorable outcome were sensitive to chemotherapy. Next, we screened the three ion genes-based signature in different molecular glioma subtypes. The signature showed a Mesenchymal subtype and wild-type IDH1 preference. Gene ontology (GO) analysis for the functional annotation of the signature showed that patients with high-risk scores tended to exhibit the increased expression of proteins associated with apoptosis, immune response, cell adhesion and motion and vasculature development. Gene Set Enrichment Analysis (GSEA) results showed that pathways associated with negative regulation of programmed cell death, cell proliferation and locomotory behavior were highly expressed in the high-risk group. These results suggest that ion channel gene expression could improve the subtype classification in gliomas at the molecular level. The findings in the present study have been validated in two independent cohorts.

A novel integrated gene coexpression analysis approach reveals a prognostic three-transcription-factor signature for glioma molecular subtypes.

BackgroundGlioma is the most common brain tumor and it has very high mortality rate due to its infiltration and heterogeneity. Precise classification of glioma subtype is essential for proper therapeutic treatment and better clinical prognosis. However, the molecular mechanism of glioma is far from clear and the classical classification methods based on traditional morphologic and histopathologic knowledge are subjective and inconsistent. Recently, classification methods based on molecular characteristics are developed with rapid progress of high throughput technology.MethodsIn the present study, we designed a novel integrated gene coexpression analysis approach, which involves differential coexpression and differential regulation analysis (DCEA and DRA), to investigate glioma prognostic biomarkers and molecular subtypes based on six glioma transcriptome data sets.ResultsWe revealed a novel three-transcription-factor signature including AHR, NFIL3 and ZNF423 for glioma molecular subtypes. This three-TF signature clusters glioma patients into three major subtypes (ZG, NG and IG subtypes) which are significantly different in patient survival as well as transcriptomic patterns. Notably, ZG subtype is featured with higher expression of ZNF423 and has better prognosis with younger age at diagnosis. NG subtype is associated with higher expression of NFIL3 and AHR, and has worse prognosis with elder age at diagnosis. According to our inferred differential networking information and previously reported signalling knowledge, we suggested testable hypotheses on the roles of AHR and NFIL3 in glioma carcinogenesis.ConclusionsWith so far the least biomarkers, our approach not only provides a novel glioma prognostic molecular classification scheme, but also helps to explore its dysregulation mechanisms. Our work is extendable to prognosis-related classification and signature identification in other cancer researches.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0315-y) contains supplementary material, which is available to authorized users.

Abstract 987: A novel cell cycle-associated lncRNA, HOXA11-AS, is transcribed from 5-prime end of HOXA transcript and a biomarker of progression in glioma

Abstract The comprehensive lncRNA expression signature in glioma remains fully unknown. We performed a high throughput microarray to detect ncRNA expression profile in 225 human glioma tissues. One novel lncRNA, HOXA11-AS with unknown function, is the antisense transcript of HOX11 gene. The results showed that HOXA11-AS was closely associated with glioma grade and poor prognosis. Multivariate cox regression analysis revealed that HOXA11-AS was an independent prognostic factor in glioblastoma multiforme patients and its expression was correlated with glioma molecular subtype of the Cancer Genome Atlas. Gene set enrichment analysis indicated that gene sets most correlated with HOXA11-AS expression involved in cell cycle progression. Overexpression of HOXA11-AS transcript promoted cell proliferation in vitro while knockdown of HOXA11-AS expression manifested a repressive function during these cellular processes via regulating cell cycle progress. The growth promoting and inhibiting effect of HOXA11-AS was also demonstrated in a xenograft mouse model. For the first time, our data confirms that HOXA11-AS is an important long non-coding RNA that primarily serves as a prognostic factor for glioma patient survival, a biomarker for identifying glioma molecular subtypes, and therapeutic target for glioma patient. Citation Format: Qixue Wang, Junxia Zhang, Yanwei Liu, Junhju Zhou, Chunsheng Kang, Lei Han. A novel cell cycle-associated lncRNA, HOXA11-AS, is transcribed from 5-prime end of HOXA transcript and a biomarker of progression in glioma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 987.

Differences of malignant proliferation and infiltrating growth related molecular changes in different pathological types of glioma

Objective To investigate the distribution of the cell cycle alteration and cell migration as well as infiltrating growth process and related genetic changes in each grade and each molecular subtype of gliomas. Methods A total of 300 glioma specimens(WHO grade Ⅱ-Ⅳ)in Chinese Glioma Genome Atlas(CGGA)were collected. Its tissue RNA and DNA were extract respectively. Sanger sequencing was used to detect the mutation status of PTEN, p53, and TERT promoter. The expression levels of MET and matrix metalloproteinase 9(MMP-9)were derived from whole-genome RNA microarray expression profile. The distribution differences of the mutations of PTEN, p53, TERT promoter and high expression of MET and MMP-9 were compared in each level and each molecular subtypes of glioma. Results (1)In WHO grade Ⅱ, Ⅲ and Ⅳ, the mutation frequencies of PTEN were 2.8%(2/72), 5.0%(1/20), and 23.0%(20/87), respectively; the mutation rates of p53 were 28.0%(21/75), 10.0%(2/20), and 20.7%(18/87), respectively; the mutation rates of TERT promoter were 30.2%(26/86), 47.1%(8/17), and 46.8%(29/62), respectively; the incidences of MET high expression were 28.9%(35/121), 33.3%(17/51), and 37.5%(48/128), respectively; and the high expression rates of MMP-9 were 3.3%(4/121), 39.2%(20/51), and 59.4%(76/128), respectively. There were significant differences in each grade of gliomas between the mutation rate of PTEN and the high expression rate of MMP-9(all P<0.01).(2)The mutation rates of PTEN in the proneural type, neural type, classical type and molecular type of mesenchymal glioma were 1.9%(1/52), 12.5%(4/32), 15.0%(3/20), and 20.0%(15/75), respectively; the mutation rates of p53 were 34.0%(18/53), 6.1%(2/33), 40.0%(8/20), and 17.1%(13/76), respectively; the mutation rates of TERT promoter were 37.5%(21/56), 34.5%(10/29), 31.3%(5/16), and 45.1%(23/51), respectively; the MET high expression rates were 25.6%(21/82), 34.5%(19/55), 28.9%(11/38), and 40.0%(38/95), respectively; the MMP-9 high expression rates were 9.8%(8/82), 1.8%(1/55), 44.7%(17/38), and 68.4%(65/95), respectively. There were significant differences in the mutation rates of PTEN and p53, and the MMP-9 high expression rates among each molecular subtype of glioma(all P<0.05). Conclusions The mutation rate of PTEN and the high expression rates of MMP-9 were associated with the malignant degree of gliomas. The differences of the mutation rates of PTEN and p53, and the high expression rates of MMP-9 in each molecular subtype of gliomas suggested that they could represent the molecular pathologic features of the specific molecular subtypes of gliomas. Key words: Glioma; Pathology; Molecular subtype; Malignant proliferation; Infiltrating growth

Targeted Therapeutics in Patients With High-Grade Gliomas: Past, Present, and Future.

High-grade gliomas remain incurable despite current therapies, which are plagued by high morbidity and mortality. Molecular categorization of glioma subtypes using mutations in isocitrate dehydrogenase 1/2 (IDH1/2), TP53, and ATRX; codeletion of chromosomes 1p and 19q; DNA methylation; and amplification of genes such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor, alpha polypeptide provides a more accurate prognostication and biologic classification than classical histopathological diagnoses, and a number of molecular markers are being incorporated in the new World Health Organization classification of gliomas. However, despite the improved understanding of the molecular subtypes of gliomas and the underlying alterations in specific signaling pathways, these observations have so far failed to result in the successful application of targeted therapies, as has occurred in other solid tumors. To date, the only targeted therapy for gliomas approved by the US Food and Drug Administration is bevacizumab, which targets vascular endothelial growth factor. EGFR remains a dominant molecular alteration in specific glioma subtypes and represents a potentially promising target, with drugs of multiple types targeting EGFR in development including vaccines, antibody drug conjugates, and chimeric antigen receptor (CAR) T cells, despite the prior failures of EGFR tyrosine kinase inhibitors. Immune therapies under investigation include checkpoint inhibitors, vaccines against tumor-associated antigens and tumor-specific antigens, pulsed dendritic cells, heat shock protein-tumor conjugates, and CAR T cells. Mutations in the IDH1/2 genes are central to gliomagenesis in a high proportion of grade II and III gliomas, and ongoing trials are examining vaccines against IDH1, small molecular inhibitors of IDH1 and IDH2, and metabolic components including NAD+ depletion to target IDH-mutated gliomas. The central role of DNA methylation in a subset of gliomas may be targetable, but better understanding of the relation between epigenetic alterations and resulting tumor biology appears necessary. Ultimately, given the prior failure of single-agent targeted therapy in high-grade gliomas, it appears that novel combinatorial therapy or targeted drugs with immunomodulatory or epigenetic approaches will likely be necessary to successfully combat these challenging tumors.

Culture conditions tailored to the cell of origin are critical for maintaining native properties and tumorigenicity of glioma cells.

Cell culture plays a pivotal role in cancer research. However, culture-induced changes in biological properties of tumor cells profoundly affect research reproducibility and translational potential. Establishing culture conditions tailored to the cancer cell of origin could resolve this problem. For glioma research, it has been previously shown that replacing serum with defined growth factors for neural stem cells (NSCs) greatly improved the retention of gene expression profile and tumorigenicity. However, among all molecular subtypes of glioma, our laboratory and others have previously shown that the oligodendrocyte precursor cell (OPC) rather than the NSC serves as the cell of origin for the proneural subtype, raising questions regarding the suitability of NSC-tailored media for culturing proneural glioma cells. OPC-originated mouse glioma cells were cultured in conditions for normal OPCs or NSCs, respectively, for multiple passages. Gene expression profiles, morphologies, tumorigenicity, and drug responsiveness of cultured cells were examined in comparison with freshly isolated tumor cells. OPC media-cultured glioma cells maintained tumorigenicity, gene expression profiles, and morphologies similar to freshly isolated tumor cells. In contrast, NSC-media cultured glioma cells gradually lost their OPC features and most tumor-initiating ability and acquired heightened sensitivity to temozolomide. To improve experimental reproducibility and translational potential of glioma research, it is important to identify the cell of origin, and subsequently apply this knowledge to establish culture conditions that allow the retention of native properties of tumor cells.

A novel cell cycle-associated lncRNA, HOXA11-AS, is transcribed from the 5-prime end of the HOXA transcript and is a biomarker of progression in glioma

The comprehensive lncRNA expression signature in glioma has not yet been fully elucidated. We performed a high-throughput microarray to detect the ncRNA expression profiles of 220 human glioma tissues. Here, we found that a novel lncRNA, HOXA11-AS, was the antisense transcript of the HOX11 gene. It was shown that HOXA11-AS was closely associated with glioma grade and poor prognosis. Multivariate Cox regression analysis revealed that HOXA11-AS was an independent prognostic factor in glioblastoma multiforme patients, and its expression was correlated with the glioma molecular subtypes of the Cancer Genome Atlas. Gene set enrichment analysis indicated that the gene sets most correlated with HOXA11-AS expression were involved in cell cycle progression. Over-expression of the HOXA11-AS transcript promoted cell proliferation in vitro, while knockdown of HOXA11-AS expression repressed cell proliferation via regulation of cell cycle progression. The growth-promoting and growth-inhibiting effects of HOXA11-AS were also demonstrated in a xenograft mouse model. Our data confirms, for the first time, that HOXA11-AS is an important long non-coding RNA that primarily serves as a prognostic factor for glioma patient survival. HOXA11-AS could serve as a biomarker for identifying glioma molecular subtypes and as therapeutic target for glioma patients.

PI3 kinase mutations and mutational load as poor prognostic markers in diffuse glioma patients.

IntroductionRecent advances in molecular diagnostics allow diffuse gliomas to be classified based on their genetic changes into distinct prognostic subtypes. However, a systematic analysis of all molecular markers has thus far not been performed; most classification schemes use a predefined and select set of genes/molecular markers. Here, we have analysed the TCGA dataset (combined glioblastoma (GBM) and lower grade glioma (LGG) datasets) to identify all prognostic genetic markers in diffuse gliomas in order to generate a comprehensive classification scheme.ResultsOf the molecular markers investigated (all genes mutated at a population frequency >1.7 % and frequent chromosomal imbalances) in the entire glioma dataset, 57 were significantly associated with overall survival. Of these, IDH1 or IDH2 mutations are associated with lowest hazard ratio, which confirms IDH as the most important prognostic marker in diffuse gliomas. Subsequent subgroup analysis largely confirms many of the currently used molecular classification schemes for diffuse gliomas (ATRX or TP53 mutations, 1p19q codeletion). Our analysis also identified PI3-kinase mutations as markers of poor prognosis in IDH-mutated + ATRX/TP53 mutated diffuse gliomas, median survival 3.7 v. 6.3 years (P = 0.02, Hazard rate (HR) 2.93, 95 % confidence interval (CI) 1.16 – 7.38). PI3-kinase mutations were also prognostic in two independent datasets. In our analysis, no additional molecular markers were identified that further refine the molecular classification of diffuse gliomas. Interestingly, these molecular classifiers do not fully explain the variability in survival observed for diffuse glioma patients. We demonstrate that tumor grade remains an important prognostic factor for overall survival in diffuse gliomas, even within molecular glioma subtypes. Tumor grade was correlated with the mutational load (the number of non-silent mutations) of the tumor: grade II diffuse gliomas harbour fewer genetic changes than grade III or IV, even within defined molecular subtypes (e.g. ATRX mutated diffuse gliomas).ConclusionWe have identified PI3K mutations as novel prognostic markers in gliomas. We also demonstrate that the mutational load is associated with tumor grade. The increase in mutational load may partially explain the increased aggressiveness of higher grade diffuse gliomas when a subset of the affected genes actively contributes to gliomagenesis and/or progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0265-4) contains supplementary material, which is available to authorized users.

GE-10 * IDENTIFICATION OF PATIENTS WITH RECURRENT GBM THAT BENEFIT FROM BEVACIZUMAB

INTRODUCTION: To assess the effects of bevacizumab (BEV) in recurrent GBMs, a three-arm randomized phase II study was initiated (the BELOB trial) comparing BEV monotherapy, CCNU monotherapy and BEV + CCNU combinationtherapy. Primary endpoint was 9 months OS and was 38% [25,51], 43% [29,57] and 59% [43,72] in the BEV, lomustine and combination arms respectively. The BELOB trial results therefore provide evidence for clinical efficacy of BEV, at least in a recurrent GBM setting and when given in combination with lomustine. However, not all patients in this trial appeared to benefit equally from the combination treatment. In this study, we therefore aimed to identify patients that benefit from BEV. METHODS: RNA expression profiling was performed using DASL (Illumina, n = 118) and RNA sequencing (n = 78) of samples. RESULTS: The DASL and RNA-seq platform both show a high concordance between technical and biological replicates and with expression profiling on snap frozen tissue samples. Sample assignment to one of seven ‘intrinsic glioma subtypes’ (IGS; molecular subtypes of glioma based on similarities in gene-expression profiles [PMID: 19920198]) was also highly similar between the different platforms. Analysis of BELOB samples shows that most are assigned to poor prognostic subtypes (IGS-18 "EGFR-amplified, classical" n = 73 and IGS-23 "mesenchymal" n = 28). Nine patients were assigned to prognostically more favourable subtypes (IGS-9 and IGS-17), but these do not explain the more favourable outcome in the BEV + CCNU arm. Patients with tumors assigned to IGS-18 show benefit from combined treatment with bevacizumab + CCNU. In contrast, tumors assigned to IGS-23 may have worse performance in this arm, though patient numbers are small. CONCLUSIONS: DASL and RNA-seq perform well on RNA isolated from FFPE tissues stored (up to 20 years) in paraffin. Specific molecular subtypes of recurrent GBM show benefit from BEV + CCNU treatment.

Abstract 4714: The association of glioma germline risk SNPs with mutation-based molecular subgroups

Abstract Background: Specific germline alterations within the TERT, EGFR, CCDC26, CDKN2A/B, PHLDB1, TP53 and RTEL1 regions are associated with development of glioma. While some of these germline variants are associated with all gliomas (e.g. rs2736100 in TERT), others are associated with a specific morphologic and/or molecular subtype. For example, the RTEL1 region variants are associated with primary glioblastoma and rs55705857 in CCDC26 is associated with 1p/19q co-deleted oligodendrogliomas and with IDH mutant astrocytic gliomas. We hypothesized that germline variants in these regions will be associated with other molecular subtypes. The Cancer Genome Atlas (TCGA) and other groups have described molecular subtypes of glioma based on acquired somatic mutation patterns. Herein, we grouped glioma patients into mutation-based molecular subgroups based on three molecular alterations and determined if germline polymorphisms are associated with these mutation-based molecular subtypes. Methods: Five molecular subtypes were defined based on combinations of the following molecular alterations: TERT promoter mutation (TERTmut vs. TERTwt), IDH1/2 mutation (IDH mut vs. IDH wt) and 1p/19q co-deletion (1p19qcodel vs. 1p19q noncodel). Germline single nucleotide polymorphisms (SNPs) were evaluated by custom Illumina array analysis of blood-derived DNA and 1K genome imputation. Results: One hundred ninety-nine gliomas (24 oligodendrogliomas, 59 mixed oligoastrocytomas, 41 grade 2-3 astrocytomas and 75 glioblastomas) had both SNP data and mutation data available in order to assign them into the five molecular subgroups. The prevalence of each of the molecular subtypes was as follows: TERTmut/IDHmut/1p19qcodel (20%), TERTmut/IDHmut/1p19qnoncodel (5%), TERTmut/IDHwt/1p19qnoncodel (32%), TERTwt/IDHmut/1p19qnondel (35%) and TERTwt/IDHwt/1p19qnoncodel (8%). The TERT SNP (rs2736100) was protective for TERTmut/IDHmut/1p19qnoncodel and TERTwt/IDHmut/1p19qnoncodel gliomas. The CCDC26 SNP (rs55705857) was strongly associated with the risk of developing TERTmut/IDHmut/1p19qcodel and TERTwt/IDHmut/1p19qnoncodel gliomas. The TP53 SNP (rs7837822) was strongly associated with risk of developing TERTmut/IDHmut/1p19qnoncodel and TERTwt/IDHmut/1p19qnoncodel gliomas. Interestingly, while one RTEL1 region SNP (rs6062297) was strongly associated with glioma risk, RTEL1 SNPs rs6010620 and rs2297440 were protective for the development of TERTmut/IDHwt/1p19qnoncodel gliomas. We are currently in the process of validating these results using the TCGA glioblastoma and low grade glioma data. Conclusions: Glioma patients can be meaningfully classified according to their acquired mutation subtype. Importantly, our results suggest that there are significant associations between germline polymorphisms and mutation-based glioma molecular subtypes. Citation Format: Jeanette E. Eckel-Passow, Thomas M. Kollmeyer, Gobinda Sarkar, Anisha Chada, Paul A. Decker, Matthew L. Kosel, Alissa A. Caron, Hugues Sicotte, Kannabiran Nandakumar, Naresh Prodduturi, Brian P. O'Neill, Daniel H. Lachance, Robert B. Jenkins. The association of glioma germline risk SNPs with mutation-based molecular subgroups. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4714. doi:10.1158/1538-7445.AM2014-4714

MUTATION-BASED MOLECULAR GLIOMA CLASSIFICATION: PREVALENCE AND ASSOCIATION WITH GERMLINE RISK SNPS

BACKGROUND: Specific germline alterations within TERT, EGFR, CCDC26, CDKN2A/B, PHLDB1, TP53 and RTEL1 are associated with development of glioma. While germline variants in TERT are associated with all gliomas, others are associated with specific morphologic and/or molecular subtypes. For example, the RTEL1 region variants are associated with primary glioblastoma and rs55705857 in CCDC26 is associated with 1p/19q co-deleted oligodendrogliomas and with IDH mutant astrocytic gliomas. We hypothesize that germline variants will be associated with specific molecular glioma subtypes recently defined by the Cancer Genome Atlas (TCGA) and other groups. In this study we grouped Mayo Clinic and TCGA high- and low-grade glioma patients into somatic mutation-defined molecular subgroups, regardless of grade, based on three molecular alterations. We then determined if germline polymorphisms are associated with these mutation-based molecular subtypes. METHODS: Five molecular subtypes were defined based on combinations of the following molecular alterations: TERT promoter mutation (TERT mut vs. TERT wt), IDH1/2 mutation (IDH mut vs. IDH wt) and 1p/19q co-deletion (1p19q codel vs. 1p19q noncodel). Germline single nucleotide polymorphisms (SNPs) were evaluated by array analysis of blood-derived DNA (Mayo) and 1K genome imputation (TCGA). RESULTS: 207 Mayo Clinic and 402 TCGA gliomas had both germline SNP and mutation data available. The acquired mutation data defined five molecular subgroups (Table). The prevalence of each of these subtypes were very similar in the Mayo and TCGA cohorts, (Table). In both the Mayo Clinic and TCGA cohorts various SNPs were associated with the development of specific subtypes (Table). Further validation using a UCSF cohort, as well as analyses of grade, subtype and survival are underway. Table Prevalence of Mutation-based Acquired Molecular Glioma Subtypes and Their Germline SNP Associations

IDENTIFICATION OF PATIENTS WITH RECURRENT GBM THAT BENEFIT FROM BEVACIZUMAB

INTRODUCTION: To assess the effects of bevacizumab (BEV) in recurrent GBMs, a three-arm randomized phase II study was initiated (the BELOB trial) comparing BEV monotherapy, CCNU monotherapy and BEV + CCNU combinationtherapy. Primary endpoint was 9 months OS and was 38% [25,51], 43% [29,57] and 59% [43,72] in the BEV, lomustine and combination arms respectively. The BELOB trial results therefore provide evidence for clinical efficacy of BEV, at least in a recurrent GBM setting and when given in combination with lomustine. However, not all patients in this trial appeared to benefit equally from the combination treatment. In this study, we therefore aimed to identify patients that benefit from BEV. METHODS: RNA expression profiling was performed using DASL (Illumina, n = 118) and RNA sequencing (n = 78) of samples. RESULTS: The DASL and RNA-seq platform both show a high concordance between technical and biological replicates and with expression profiling on snap frozen tissue samples. Sample assignment to one of seven ‘intrinsic glioma subtypes’ (IGS; molecular subtypes of glioma based on similarities in gene-expression profiles [PMID: 19920198]) was also highly similar between the different platforms. Analysis of BELOB samples shows that most are assigned to poor prognostic subtypes (IGS-18 EGFR-amplified, classical n = 73 and IGS-23 mesenchymal n = 28). Nine patients were assigned to prognostically more favourable subtypes (IGS-9 and IGS-17), but these do not explain the more favourable outcome in the BEV + CCNU arm. Patients with tumors assigned to IGS-18 show benefit from combined treatment with bevacizumab + CCNU. In contrast, tumors assigned to IGS-23 may have worse performance in this arm, though patient numbers are small. CONCLUSIONS: DASL and RNA-seq perform well on RNA isolated from FFPE tissues stored (up to 20 years) in paraffin. Specific molecular subtypes of recurrent GBM show benefit from BEV + CCNU treatment.

HOTAIR, a cell cycle–associated long noncoding RNA and a strong predictor of survival, is preferentially expressed in classical and mesenchymal glioma

Long noncoding RNA Hox transcript antisense intergenic RNA (HOTAIR) has been characterized as a negative prognostic factor in breast and colon cancer patients. The clinical significance and function of HOTAIR in glioma remains unclear. We analyzed the clinical significance of HOTAIR in 3 different glioma cohorts with gene expression data, including correlation with tumor grade, prognosis, and molecular subtype. The function of HOTAIR in glioma was explored by performing gene set enrichment analysis and in vitro and in vivo experiments. HOTAIR expression was closely associated with glioma grade and poor prognosis. Multivariate Cox regression analysis revealed that HOTAIR was an independent prognostic factor in glioblastoma multiforme patients. HOTAIR expression correlated with glioma molecular subtype, including those of The Cancer Genome Atlas. HOTAIR was preferentially expressed in the classical and mesenchymal subtypes compared with the neural and proneural subtypes. A gene set enrichment analysis designed to show gene set differences between patients with high and low HOTAIR expression indicated that HOTAIR expression was associated with gene sets involved in cell cycle progression. HOTAIR reduction induced colony formation suppression, cell cycle G0/G1 arrest, and orthotopic tumor growth inhibition. Our data establish that HOTAIR is an important long noncoding RNA that primarily serves as a prognostic factor for glioma patient survival, as well as a biomarker for identifying glioma molecular subtypes, a critical regulator of cell cycle progression.

Predictive biomarkers in adult gliomas

This review summarizes recent studies on the predictive value of molecular markers in adult gliomas, including 1p/19q codeletion, MGMT methylation, IDH mutation and markers identified using omics and next-generation sequencing studies. The long-term results of the Radiation Therapy Oncology Group and European Organization for Research and Treatment of Cancer trials in anaplastic oligodendroglial glioma have shown that the 1p/19q codeletion predicts an overall survival benefit from early PCV (procarbazine CCNU vincristine) chemotherapy. This benefit can also be predicted using gene expression-based molecular subtypes of gliomas while the predictive value of the IDH mutation in this context requires further study. In elderly patients with glioblastoma, the analysis of MGMT methylation status in two phase III trials suggests that this alteration may guide treatment decisions; however, this finding still needs confirmation in prospective studies. Omics and next-generation sequencing studies have identified additional potential predictive markers. In particular, IDH mutations, BRAF V600E mutations and FGFR gene fusions might predict efficacy of therapies targeted against these alterations. Currently, the 1p/19q codeletion is the only well established predictive marker with clinical utility. However, it is likely that other molecular markers such as MGMT methylation, IDH mutation and those identified using omics and next-generation sequencing studies will further guide treatment decisions in adult gliomas.

Raman spectroscopy can discriminate distinct glioma subtypes as defined by RNA expression profiling

Raman spectroscopy is a molecular spectroscopic technique that can measure the molecular composition of tissue samples within seconds without any extraction processes or dyes. In microbiology, Raman spectroscopy is used to identify bacteriae. In glioblastoma tissue, it was reported that necrosis, normal brain and tumor can be discriminated using Raman spectroscopy. Therefore, we hypothesized that Raman spectroscopy could discriminate glioblastoma tissue from different glioma subtypes defined by RNA expression profiling.We analyzed 20 glioma samples from two distinct molecular subtypes. Both subtypes consisted of glioblastoma samples showing a variety in glioma grading and typing.The Raman spectroscopic results could be grouped in two distinct clusters in an unsupervised cluster analysis. Further analysis of these clusters showed that they were fully congruent with the two clusters as defined by RNA expression profiling.Conclusion: our results demonstrate that Raman spectroscopy can discriminate between different molecular subtypes of glioma and, therefore, may prove to be a valuable tool in in vitro cancer research. Copyright © 2013 John Wiley &amp; Sons, Ltd.

Molecular subtypes of glioma identified by genome‐wide methylation profiling

Recent studies have indicated a prognostic role for genome-wide methylation in gliomas: Tumors that show an overall increase in DNA methylation at CpG sites (CIMP+; CpG island methylator phenotype) have a more favorable prognosis than CIMP- gliomas. Here, we have determined whether methylation profiling can identify more and clinically relevant molecular subtypes of glioma by performing genome-wide methylation profiling on 138 glial brain tumors of all histological diagnosis. Hopach (Hierarchical ordered partitioning and collapsing hybrid) clustering using the 1,000 most variable CpGs identified three distinct glioma subtypes (C+(1p19q), C+(wt), and C-) and one adult brain subtype. All "C+(1p19q) " and "C+(wt)" tumors were CIMP+ whereas most (50/54) "C-" tumors were CIMP-. The C- subtype gliomas contained many glioblastomas and all pilocytic astrocytomas. 1p19q LOH was frequent in the C+(1p19q) subtype. Other genetic changes (IDH1 mutation and EGFR amplification) and gene-expression based molecular subtypes also segregated in distinct methylation subtypes, demonstrating that these subtypes are also genetically distinct. Each subtype was associated with its own prognosis: median survival for C-, C+(1p19q), and C+(wt) tumors was 1.18, 5.00, and 2.62 years, respectively. The prognostic value of these methylation subtypes was validated on an external dataset from the TCGA. Analysis of recurrences of 14 primary tumors samples indicates that shifts between some C+(wt) and C+(1p/19q) tumors can occur between the primary and recurrent tumor, but CIMP status remained stable. Our data demonstrate that methylation profiling identifies at least three prognostically relevant subtypes of glioma that can aid diagnosis and potentially guide treatment for patients.

Intrinsic Molecular Subtypes of Glioma Are Prognostic and Predict Benefit From Adjuvant Procarbazine, Lomustine, and Vincristine Chemotherapy in Combination With Other Prognostic Factors in Anaplastic Oligodendroglial Brain Tumors: A Report From EORTC Study 26951

Intrinsic glioma subtypes (IGSs) are molecularly similar tumors that can be identified based on unsupervised gene expression analysis. Here, we have evaluated the clinical relevance of these subtypes within European Organisation for Research and Treatment of Cancer (EORTC) 26951, a randomized phase III clinical trial investigating adjuvant procarbazine, lomustine, and vincristine (PCV) chemotherapy in anaplastic oligodendroglial tumors. Our study includes gene expression profiles of formalin-fixed, paraffin-embedded (FFPE) clinical trial samples. Gene expression profiling was performed in 140 samples, 47 fresh frozen samples and 93 FFPE samples, on HU133_Plus_2.0 and HuEx_1.0_st arrays, respectively. All previously identified six IGSs are present in EORTC 26951. This confirms that different molecular subtypes are present within a well-defined histologic subtype. Intrinsic subtypes are highly prognostic for overall survival (OS) and progression-free survival (PFS). They are prognostic for PFS independent of clinical (age, performance status, and tumor location), molecular (1p/19q loss of heterozygosity [LOH], IDH1 mutation, and MGMT methylation), and histologic parameters. Combining known molecular (1p/19q LOH, IDH1) prognostic parameters with intrinsic subtypes improves outcome prediction (proportion of explained variation, 30% v 23% for each individual group of factors). Specific genetic changes (IDH1, 1p/19q LOH, and EGFR amplification) segregate into different subtypes. We identified one subtype, IGS-9 (characterized by a high percentage of 1p/19q LOH and IDH1 mutations), that especially benefits from PCV chemotherapy. Median OS in this subtype was 5.5 years after radiotherapy (RT) alone versus 12.8 years after RT/PCV (P = .0349; hazard ratio, 2.18; 95% CI, 1.06 to 4.50). Intrinsic subtypes are highly prognostic in EORTC 26951 and improve outcome prediction when combined with other prognostic factors. Tumors assigned to IGS-9 benefit from adjuvant PCV.

Biology, genetics and imaging of glial cell tumours

Despite advances in therapy, gliomas remain associated with poor prognosis. Clinical advances will be achieved through molecularly targeted biological therapies, for which knowledge of molecular genetic and gene expression characteristics in relation to histopathology and in vivo imaging are essential. Recent research supports the molecular classification of gliomas based on genetic alterations or gene expression profiles, and imaging data supports the concept that molecular subtypes of glioma may be distinguished through non-invasive anatomical, physiological and metabolic imaging techniques, suggesting differences in the baseline biology of genetic subtypes of infiltrating glioma. Furthermore, MRI signatures are now being associated with complex gene expression profiles and cellular signalling pathways through genome-wide microarray studies using samples obtained by image guidance which may be co-registered with clinical imaging. In this review we describe the pathobiology, molecular pathogenesis, stem cells and imaging characteristics of gliomas with emphasis on astrocytomas and oligodendroglial neoplasms.