Molecular And Cellular Heterogeneity: The Hallmark Of .
Neurosurg Focus 37 (6):E11, 2014 AANS, 2014Molecular and cellular heterogeneity: the hallmark ofglioblastomaDiane J. Aum, B.S.,1 David H. Kim, M.S.,1 Thomas L. Beaumont, M.D., Ph.D.,1Eric C. Leuthardt, M.D.,1,6–9 Gavin P. Dunn, M.D., Ph.D.,1,4,5,7and Albert H. Kim, M.D., Ph.D.1–3,6,7Departments of 1Neurological Surgery, 2Neurology, 3Developmental Biology, 4Pathology and Immunology,8Biomedical Engineering, and 9Mechanical Engineering and Material Sciences; 5Center for HumanImmunology and Immunotherapy Programs; 6Center for Innovation in Neuroscience and Technology; and7Siteman Cancer Center, Washington University School of Medicine in St. Louis, MissouriThere has been increasing awareness that glioblastoma, which may seem histopathologically similar acrossmany tumors, actually represents a group of molecularly distinct tumors. Emerging evidence suggests that cells evenwithin the same tumor exhibit wide-ranging molecular diversity. Parallel to the discoveries of molecular heterogeneity among tumors and their individual cells, intense investigation of the cellular biology of glioblastoma has revealedthat not all cancer cells within a given tumor behave the same. The identification of a subpopulation of brain tumorcells termed “glioblastoma cancer stem cells” or “tumor-initiating cells” has implications for the management of glioblastoma. This focused review will therefore summarize emerging concepts on the molecular and cellular heterogeneity of glioblastoma and emphasize that we should begin to consider each individual glioblastoma to be an ensembleof molecularly distinct subclones that reflect a spectrum of dynamic cell OCUS14521)Key WordsG glioblastoma is the most common primary malignant brain tumor in adults and continues to portend poor prognosis despite decades of research.Even with aggressive resection followed by concomitantchemotherapy and radiation, there is a high recurrencerate with median survival of less than 15 months.58 Indeed, fewer than 5% of patients survive 5 years.45 Extensive investigation of the cellular and molecular biologyof glioblastoma over the last decade has identified severalhistopathological and chromosomal hallmarks that haveenhanced diagnosis and clinical stratification without asignificant improvement in overall outcome. Nevertheless, a deepening focus on the molecular characteristicsof glioblastoma has revealed that this histopathological term comprises a range of diseases, some of whichhave divergent natural histories. Indeed, it is becomingclear that malignant glioma exhibits striking cellular andmolecular heterogeneity not only across but also withinglioblastomas. In fact, a patient with glioblastoma likelylioblastomaAbbreviations used in this paper: G-CIMP glioma CpG islandmethylator phenotype; H3K27Ac histone H3 Lysine27 acetylation; RTK receptor tyrosine kinase; SCNA somatic copy numberalteration; TCGA The Cancer Genome Atlas; TIC tumor-initiating cell; 2-HG 2-hydroxyglutarate.Neurosurg Focus / Volume 37 / December 2014glioma genomic heterogeneity IDH1harbors several different diseases within the same tumorsuch that each tumor is actually a complex and dynamicconstellation of cellular and molecular changes that contribute to disease pathobiology.Cellular and molecular heterogeneity is not unique toglioblastoma and has been extensively studied in severalother solid tumors. Next-generation sequencing of subregions and even single cells within breast,42 pancreas,12,68prostate,27 and renal25 cancer have demonstrated extensive regional clonality within primary tumors, where onlyone-third of mutations, known as founding mutations,occur ubiquitously in all cells. Remarkably, even minorsubclones from a primary tumor may become treatmentrefractory clones that dominate the recurrence.27 Thisconcept of clonal evolution has been firmly establishedin acute myeloid leukemia, where deep sequencing hasrevealed that subclones representing as little as 5% ofthe total tumor cells can survive treatment, acquire newmutations, and lead to relapse.21 In the related context ofmetastatic disease, deep sequencing of driver mutationsidentified in genomic studies of medulloblastoma revealed that while distinct metastatic clones within a givenpatient are genetically similar, they are divergent from theprimary tumor, suggesting subclonal enrichment and/orevolution.44,661Unauthenticated Downloaded 05/06/21 02:39 AM UTC
D. J. Aum et al.It is clear from these studies in other cancers and aquickly accumulating body of literature in glioblastomathat an improved understanding of glioblastoma heterogeneity has important implications not only for the direction of future scientific investigation, but also for clinicaldiagnosis and patient management. Herein, we review thesalient themes of tumor heterogeneity on the molecularand cellular levels and highlight the critical studies germane to neurosurgeons and neuro-oncologists that aredriving our growing understanding that diversity and heterogeneity in malignant glioma are the rules, rather thanthe exceptions (Table 1).Molecular HeterogeneityMolecular Classification: Diversity Across Tumors at theRNA LevelTranscriptomic Subtypes. Commitment by cancerbiologists to understanding the genomic basis of glioblastoma has yielded ever higher-resolution pictures ofthe types of alterations that glioblastomas harbor. Thesestudies have been central to the ongoing revolution incancer genomics that is revealing the molecular originsof every cancer type and establishing a new disease taxonomy founded on shared and/or recurrent genomic lesions.40 Broadly speaking, this sea change in cancer genomics has been possible due to our ability to performgenome-wide profiling of tumor DNA and RNA in asystematic fashion.23 In the case of glioblastoma, perhaps one of the greatest influences in our early understanding of glioma heterogeneity was the application oftranscriptional profiling to brain tumor classification.10,30Transcriptional profiling, pioneered by Golub et al.26 andAlizadeh and colleagues,1 has provided powerful evidence demonstrating the striking heterogeneity that existsamong glioblastomas arising in different patients. Amongmany, 2 studies in particular have provided our workingfoundation of glioblastoma subtypes defined by transcriptomic structure. Phillips et al. identified 3 transcriptionalsubtypes—proneural, mesenchymal, and proliferative—based on expressed genes that were most strongly correlated with survival.49 Complementing this work, Verhaak et al. employed unsupervised clustering approachesto classify 200 glioblastomas into 4 subtypes—proneural,mesenchymal, classical, and neural.63 As discussed below,these subtypes also harbor distinct DNA alterations, further emphasizing intertumoral heterogeneity and distinctpathways of tumor evolution. Importantly, although thespecific number of subtypes defined by these 2 studiesdiffers, the proneural and mesenchymal classificationsappear to be the most robust and concordant.30 Specifically, both studies identified expression of proneural genessuch as DLL3 and OLIG2 as well as mesenchymal classexpression of CD40 and CHI3L1/YKL-40. Further workhas attempted to develop prognostic assays based on alimited set of genes that correlates with survival, whichis compelling but has not yet yielded completely overlapping gene sets across studies.9,19 Nevertheless, the study ofgene expression programs and signatures has been pivotalto the evolution of a conceptual framework that glioblas2toma development is variable from patient to patient—i.e.,the “subtype” heuristic—and surely will continue to drivekey biological insights. Although beyond the scope ofthis review, there has also been a surge of interest in theepigenetic landscape of glioblastoma, and further understanding of these structural epigenetic phenotypes willlikely yield similarly important insights into intertumoralheterogeneity and natural history.11,59Molecular Classification: Diversity Across Tumors at theDNA LevelThe landmark glioblastoma expression mRNA profiling studies have been complemented by equally pivotalDNA profiling studies. To date, the glioblastoma genomeis one of the most comprehensively annotated among allcancer types, and the extensive work in this area has highlighted many of the recurrent pathways that are altered inthese tumors. At the DNA level, glioblastoma is similarto other cancers in that there are gains and losses of generegions and even whole chromosomes; these somaticcopy number alterations (SCNAs) have been cataloged bya number of groups6,48,52,65 using sophisticated computational methodologies such as Genomic Identification ofSignificant Targets in Cancer (GISTIC)6,41 and GenomicTopography Scan.65 Together, these studies identifiedcommonly amplified genes such as EGFR, MET, PDGFRA, MDM2, PIK3CA, CDK4, and CDK6 as commonlydeleted genes including CDKN2A/B, PTEN, and RB1.In addition to clarifying the gains and losses of DNAsegments, more recent work has focused on the alterationsin glioblastoma at the single nucleotide level using sequencing approaches. There is no question that the application of next-generation sequencing approaches to the studyof cancer has fueled a punctuated leap in our understanding of the genomic landscape across all cancer types. Workfrom the Vogelstein group46 and The Cancer Genome Atlas(TCGA)13 first demonstrated the types and frequencies ofmutations seen in glioma, albeit using distinct approaches.While both provided significantly overlapping information,Parsons et al.46 identified recurrent mutations in the IDH1gene, which was a seismic finding to be discussed furtherbelow, while work from the TCGA paper also provided aproof-of-principle showing that systematic, multiplatformprofiling of tumor types was possible. These studies werea prelude to the comprehensive, nearly encyclopedic anthology of the glioblastoma genome published by TCGAworking group in 2013.11 This study, analyzing more than500 glioblastomas, integrated information from sequencing, SCNA analysis, transcriptomic analysis, epigeneticprofiling, and rearrangement studies to provide a breathtaking molecular picture of these tumors. Recurrent mutations were identified in genes such as PTEN (31%), TP53(29%), EGFR (26%), PIK3R1 (11%), PIK3CA (11%), andIDH1 (5%). In addition, TERT promoter mutations wereidentified in the majority of tumors assessed by whole genome sequencing, corroborating previous findings.34 Novelmutations in genes such as LZTR, SPTA1, and TCHH werealso identified.Most importantly, the systematic TCGA profiling ofa large number of tumors enabled high-level integrationof structural genomic information that provided a moreNeurosurg Focus / Volume 37 / December 2014Unauthenticated Downloaded 05/06/21 02:39 AM UTC
Intertumoral HeterogeneityGene expression profiling identifies prognostic subclasses of high-grade gliomas that resemble stages in neurogenesis.Proneural class was enriched in neuronal markers & exhibited increased survival.Poor prognosis subclasses exhibited markers of proliferation or angiogenesis & mesenchyme.Distinct gene expression signatures & aberrations in EGFR, NF1, & PDGFRA/IDH1 define classic, mesenchymal, & proneuralsubtypes, respectively.TranscriptionalStrong relationship observed between subtypes & different neural lineages.subtypesPCR sequencing of 22 glioblastoma samples revealed the following mutations:TP53 pathway (64%): TP53, MDM2, MDM4RB1 pathway (68%): RB1, CDK4, CDKN2API3K pathway (50%): PIK3CA, PIK3R1, PTEN, IRS1Genomic differencesHighfrequency of IDH1 alterations in secondary glioblastomas & younger patients.across tumorsMutant isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with reductionof NADP to NADPH, & the production of 2HG.Elevated levels of 2HG are found in malignant gliomas harboring IDH1 mutations but not in wild type.IDH1 status is prognostic for overall survival in high-grade gliomas.IDH1 mutation IDH1 mutant tumors appear to have distinct radiographic characteristics & the metabolite 2HG can be detected by MR spectroscopy.All IDH1 mutant low-grade gliomas demonstrated IDH1 mutations at recurrence, despite substantial differences in other tumorassociated genes, suggesting this alteration occurs in the tumor cell of origin.IDH1 mutation was independently associated w/ complete resection of enhancing disease, indicating that IDH1 mutant gliomasare more amenable to maximal resection.Multiple RTK amplifications present in distinct intermingled subpopulations of tumor cells.RTKsTCGA showed 34/463 cases w/ independent focal amplification of 2 or more RTKs, most commonly PDGFRA & EGFR.Tumor cell subpopulations amplified for one RTK predominated over cells amplified for both.Over half of glioblastoma samples overexpressed EGFR (50–70% of these expressed EGFRvIII).Glioblastoma with EGFRvIII mutation always harbored EGFR amplification, suggesting EGFR amplification precedes EGFR mutation.Protein level heterogene- EGFRvIII muta- Wild-type EGFR distribution was homogeneous, & EGFRvIII distribution was mainly diffusely scattered.tionity across tumorsEGFRvIII cells express IL-6 &/or cytokines that activate gp130, which in turn activates wild-type EGFR in neighboring cells, causing accelerated proliferation.IL-6, LIF, or gp130 ablation uncoupled this EGFRvIII cell-driven paracrine mechanism & potently attenuated tumor growth.Tumor cell lines, xenotransplants, & primary tumors showed multiple concomitantly activated RTKs.Receptor coactiCombinations of RTK inhibitors &/or RNA interference, but not single agents, decreased signaling, cell survival, & anchoragevationindependent growth.TABLE 1: Molecular and cellular heterogeneity in glioblastoma*Neurosurg Focus / Volume 37 / December 2014(continued)Stommel et al., 2007Inda et al., 2010Nishikawa et al., 2004Snuderl et al., 2011Szerlip et al., 2012Beiko et al., 2014Hartmann et al., 2010,& Lai et al., 2011Johnson et al., 2014Dang et al., 2009Parsons et al., 2008Verhaak et al., 2010Phillips et al., 2006Molecular and cellular heterogeneity in glioblastoma3Unauthenticated Downloaded 05/06/21 02:39 AM UTC
4The IDH1 Story: Not all Gliomas Are Created Equal* IL interleukin; LIF leukemia inhibitory factor; PCR polymerase chain reaction; 2HG 2-hydroxyglutarate.TIC populationRTKsgranular picture of intertumor heterogeneity. Specifically,it was possible to integrate DNA-based alterations withRNA-defined subtypes. For instance, proneural tumorsoften harbor mutations in IDH1, TP53, and ATRX andcan exhibit the glioma CpG island methylator phenotype(G-CIMP); IDH1 wild-type tumors tend to be G-CIMPnegative and harbor PDGFRA/CDK4 amplifications. Incontrast, mesenchymal tumors are IDH1 wild type butharbor NF1 mutations. In addition, classical tumors morefrequently harbor EGFR mutations, including the EGFRvIII intragenic deletion, in the setting of high-level EGFRamplification. Additional work in this effort provided information on novel gene rearrangements and epigeneticlandscapes as well. Taken together, this study providedhigh-resolution analysis of the molecular diversity observed across different glioblastoma tumors. This information can be accessed either through TCGA (http://cancergenome.nih.gov) or through independent portals thatenable rapid accession of the data in visual format (BroadInstitute TCGA portal at http://www. broadinstitute.org/tcga and the Memorial Sloan–Kettering Cancer CentercBio Portal at http://www.cbioportal.org).Suvà et al., 2014Soda et al., 2011, &Turcan et al., 2012Singh et al., 2004Bhat et al., 2013Patel et al., 2014Transcriptional differences btwn cellsSingle-cell whole genome sequencing analysis of multiple EGFR aberrations w/in the same tumor identified different clonal events.EGFR truncation variants segregated into nonoverlapping subclonal populations.Glioblastoma subtype classifiers were variably expressed across individual cells w/in a single tumor.A spectrum of “stemness”-related expression states was observed w/in a tumor.Proneural classification was associated w/ increased overall survival.RTK-amplified subpopulations share a common early precursor w/ genetic alterations such as homozygous deletion of CDKN2Aor TP53 mutation.A CD133 cell subpopulation from human brain tumors exhibited stem cell–like properties & initiated tumors in vivo.Human glioma sphere cultures cluster into 2 dominant transcriptional subtypes: proneural & mesenchymal.Conversion from proneural to mesenchymal TICs can occur in a TNFα & NF-κB dependent manner.The mesenchymal TIC signature, CD44 expression, and NF-κB activation correlate w/ relative radioresistance & shorter survival.A core set of neurodevelopmental transcription factors (POU3F2, SOX2, SALL2, & OLIG2) coordinately activate an epigeneticprogram that defines TIC identity.These master regulatory transcription factors are sufficient to fully reprogram differentiated glioblastoma cells to ‘‘induced’’ TICs.Structural DNA alterations btwn cellsTABLE 1: Molecular and cellular heterogeneity in glioblastoma* (continued)Intratumoral HeterogeneityFrancis et al., 2014D. J. Aum et al.If there is a single mutation that underscores theidea that not all glioblastomas are created equally, it isthe mutation found in the IDH1 gene.22,23 IDH1 encodesisocitrate dehydrogenase 1, a cytoplasmic enzyme thatcatalyzes the oxidative decarboxylation of isocitrate toa-ketoglutarate and NAD to NADPH.50 Recurrent mutations in IDH1 were first identified by Parsons et al. in2008 in a study in which 5 of 22 tumors in the study’s discovery set harbored a single mutation encoding the IDH1R132H mutant protein. Extensive work by many groupshas since demonstrated how important the distinctionbetween “IDH1 mutant” and “IDH1 wild type” may be.In brain tumors, IDH1 mutations are typically R132H(90%), although other substitutions at position R132 arealso found.69 IDH1 mutations are identified in 60%–80%of Grade II and III astrocytomas, oligodendrogliomas,and oligoastrocytomas.23,28,29 Whereas 5%–6% of primary glioblastomas harbor IDH1 mutations, the majorityof secondary glioblastomas carry this mutation. Similarmutations in the IDH2 gene often seen in acute myeloidleukemia are rarely found in glioma. Interestingly, mutations in the IDH genes have also been identified in cholangiocarcinomas and cartilaginous tumors.23The foundation of all efforts to understand IDH1 mutant protein biology is the work of Dang and colleagues.20This study used a liquid chromatography/mass spectrometry metabolomics approach to discover that IDH1R132H mutant proteins acquire a neomorphic functionthat leads to the production of high levels of the “R”enantiomer of 2-hydroxyglutarate (2-HG) that are neverseen at comparable levels in an IDH1 wild-type settingand can be detected using MR spectroscopy.2 Becausetumor cells typically harbor one copy of IDH1 mutationand do not exhibit loss of heterozygosity at that locus,these mutations are likely gain of function in nature. Significant effort is underway to understand the mechanisticunderpinnings of the IDH1 mutant state. Although 2-HGNeurosurg Focus / Volume 37 / December 2014Unauthenticated Downloaded 05/06/21 02:39 AM UTC
Molecular and cellular heterogeneity in glioblastomahas been shown to inhibit the a-ketoglutarate–dependentsuperfamily of dioxygenases affecting histone and DNAmethylation,18,67 likely contributes to the G-CIMP phenotype,62 and also alters HIF-1a biology,23,35 further work isnecessary to clarify the causal mechanistic basis of mutant IDH1 biology.Accumulating evidence suggests that IDH1 mutantand IDH1 wild-type tumors should perhaps be considered different tumors, albeit with similar histopathology.Patients with IDH1 mutant tumors are nearly 2 decadesyounger than patients with IDH1 wild-type tumors andexhibit an overall survival that can be up to 3-fold greaterthan p
constellation of cellular and molecular changes that con-tribute to disease pathobiology. Cellular and molecular heterogeneity is not unique to glioblastoma and has been extensively studied in several other soli
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