ABSTRACT
Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.
Subject(s)
Central Nervous System Neoplasms/genetics , Central Nervous System Neoplasms/pathology , DNA Methylation , Neuroectodermal Tumors/genetics , Neuroectodermal Tumors/pathology , Amino Acid Sequence , Central Nervous System Neoplasms/classification , Central Nervous System Neoplasms/diagnosis , Child , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Molecular Sequence Data , Neuroectodermal Tumors/classification , Neuroectodermal Tumors/diagnosis , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Repressor Proteins/chemistry , Repressor Proteins/genetics , Signal Transduction , Trans-Activators , Tumor Suppressor Proteins/geneticsABSTRACT
Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.
Subject(s)
MicroRNAs/genetics , Neoplasms, Germ Cell and Embryonal/genetics , DEAD-box RNA Helicases/genetics , DNA Topoisomerases, Type I/genetics , Humans , Mutation , Neoplasms, Germ Cell and Embryonal/diagnosis , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/genetics , Polymorphism, Single Nucleotide , RNA, Long Noncoding , Recurrence , Ribonuclease III/geneticsABSTRACT
Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1). Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat.
Subject(s)
Enhancer Elements, Genetic/genetics , Ependymoma/drug therapy , Ependymoma/genetics , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks/genetics , Molecular Targeted Therapy , Oncogenes/genetics , Transcription Factors/metabolism , Animals , Base Sequence , Ependymoma/classification , Ependymoma/pathology , Female , Humans , Mice , Precision Medicine , RNA Interference , Xenograft Model Antitumor AssaysABSTRACT
Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.
Subject(s)
DNA Mutational Analysis , Genome, Human/genetics , Medulloblastoma/classification , Medulloblastoma/genetics , Whole Genome Sequencing , Carcinogenesis/genetics , Carrier Proteins/genetics , Cohort Studies , DNA Methylation , Datasets as Topic , Epistasis, Genetic , Genomics , Humans , Molecular Targeted Therapy , Muscle Proteins/genetics , Mutation , Oncogenes/genetics , Transcription Factors/genetics , Wnt Proteins/geneticsABSTRACT
Medulloblastoma is the primary malignant embryonic tumor of the cerebellum and the most common malignant tumor of childhood, accounting up to 25% of all CNS tumors in children, but is extremely rare in adults. Despite the fact that medulloblastomas are one of the most malignant human tumors, it is worthy to note that a great breakthrough has been achieved in our understanding of oncogenesis and the development of real methods of treatment. The main objective of surgical treatment is a maximum resection of tumor with minimal impairment of neurological functions, in order to reduce the volume, remove tumor tissue, get the biopsy, and restore the cerebrospinal fluid flow. The progress of surgical techniques (using a microscope, ultrasound suction), anesthesiology, and intensive care has significantly decreased surgical mortality and increased radicality of tumor removal. Postoperative mortality is less than one percent in most studies, while neurological complications have been reported between 5-10%. Radiotherapy is the main method of treatment in patients older than 3 years, which dramatically improved the recurrence-free survival. Nevertheless, the radiation therapy without systemic chemotherapy leads to a high risk of systemic metastases. After the role of chemotherapy was statistically proven, investigations of the optimal combination of different chemotherapy regimens continued around the world. Currently, 80% of patients can already be cured, however, the quality of life of patients in the long-term period remains quite low, which depends on many factors including endocrinological, cognitive, neurological, and otoneurologic aspects. Thus, the main strategic goal of the development of neuro-oncology is to reduce the doses of radiation therapy to the CNS and the main task of international research is to optimize existing protocols and develop fundamentally new ones based on molecular genetic research in order to improve the quality of life.
Subject(s)
Cerebellar Neoplasms , Medulloblastoma , Child , Humans , Adult , Medulloblastoma/therapy , Quality of Life , Combined Modality Therapy , Antineoplastic Combined Chemotherapy Protocols , Cerebellar Neoplasms/drug therapy , Cerebellar Neoplasms/radiotherapyABSTRACT
Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.
Subject(s)
Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/pathology , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Neoplastic/genetics , Medulloblastoma/classification , Medulloblastoma/pathology , Transcription Factors/metabolism , Animals , Cerebellar Neoplasms/classification , Female , Gene Regulatory Networks/genetics , Genes, Neoplasm/genetics , Genes, Reporter/genetics , Humans , Male , Medulloblastoma/genetics , Mice , Reproducibility of Results , Zebrafish/geneticsABSTRACT
Low-grade gliomas (LGGs) are the most common childhood brain tumor in the general population and in individuals with the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Surgical biopsy is rarely performed prior to treatment in the setting of NF1, resulting in a paucity of tumor genomic information. To define the molecular landscape of NF1-associated LGGs (NF1-LGG), we integrated clinical data, histological diagnoses, and multi-level genetic/genomic analyses on 70 individuals from 25 centers worldwide. Whereas, most tumors harbored bi-allelic NF1 inactivation as the only genetic abnormality, 11% had additional mutations. Moreover, tumors classified as non-pilocytic astrocytoma based on DNA methylation analysis were significantly more likely to harbor these additional mutations. The most common secondary alteration was FGFR1 mutation, which conferred an additional growth advantage in multiple complementary experimental murine Nf1 models. Taken together, this comprehensive characterization has important implications for the management of children with NF1-LGG, distinct from their sporadic counterparts.
Subject(s)
Brain Neoplasms/genetics , Glioma/genetics , Neurofibromatosis 1/complications , Adolescent , Animals , Child , Child, Preschool , Female , Humans , Infant , Male , Mice , MutationABSTRACT
Medulloblastoma, a common pediatric malignant central nervous system tumour, represent a small proportion of brain tumours in adults. Previously it has been shown that in adults, Sonic Hedgehog (SHH)-activated tumours predominate, with Wingless-type (WNT) and Group 4 being less common, but molecular risk stratification remains a challenge. We performed an integrated analysis consisting of genome-wide methylation profiling, copy number profiling, somatic nucleotide variants and correlation of clinical variables across a cohort of 191 adult medulloblastoma cases identified through the Medulloblastoma Advanced Genomics International Consortium. We identified 30 WNT, 112 SHH, 6 Group 3, and 41 Group 4 tumours. Patients with SHH tumours were significantly older at diagnosis compared to other subgroups (p < 0.0001). Five-year progression-free survival (PFS) for WNT, SHH, Group 3, and Group 4 tumours was 64.4 (48.0-86.5), 61.9% (51.6-74.2), 80.0% (95% CI 51.6-100.0), and 44.9% (95% CI 28.6-70.7), respectively (p = 0.06). None of the clinical variables (age, sex, metastatic status, extent of resection, chemotherapy, radiotherapy) were associated with subgroup-specific PFS. Survival among patients with SHH tumours was significantly worse for cases with chromosome 3p loss (HR 2.9, 95% CI 1.1-7.6; p = 0.02), chromosome 10q loss (HR 4.6, 95% CI 2.3-9.4; p < 0.0001), chromosome 17p loss (HR 2.3, 95% CI 1.1-4.8; p = 0.02), and PTCH1 mutations (HR 2.6, 95% CI 1.1-6.2; p = 0.04). The prognostic significance of 3p loss and 10q loss persisted in multivariable regression models. For Group 4 tumours, chromosome 8 loss was strongly associated with improved survival, which was validated in a non-overlapping cohort (combined cohort HR 0.2, 95% CI 0.1-0.7; p = 0.007). Unlike in pediatric medulloblastoma, whole chromosome 11 loss in Group 4 and chromosome 14q loss in SHH was not associated with improved survival, where MYCN, GLI2 and MYC amplification were rare. In sum, we report unique subgroup-specific cytogenetic features of adult medulloblastoma, which are distinct from those in younger patients, and correlate with survival disparities. Our findings suggest that clinical trials that incorporate new strategies tailored to high-risk adult medulloblastoma patients are urgently needed.
Subject(s)
Cerebellar Neoplasms/genetics , Medulloblastoma/genetics , Adolescent , Adult , Biomarkers, Tumor/genetics , Cerebellar Neoplasms/mortality , Cerebellar Neoplasms/pathology , Cohort Studies , Female , Humans , Male , Medulloblastoma/mortality , Medulloblastoma/pathology , Progression-Free Survival , Risk Factors , Young AdultABSTRACT
Medulloblastoma with extensive nodularity (MBEN) is one of the few central nervous system (CNS) tumor entities occurring in infants which is traditionally associated with good to excellent prognosis. Some MBEN, however, have been reported with an unfavorable clinical course. We performed an integrated DNA/RNA-based molecular analysis of a multi-institutional MBEN cohort (n = 41) to identify molecular events which might be responsible for variability in patients' clinical outcomes. RNA sequencing analysis of this MBEN cohort disclosed two clear transcriptome clusters (TCL) of these CNS tumors: "TCL1 MBEN" and "TCL2 MBEN" which were associated with various gene expression signatures, mutational landscapes and, importantly, prognosis. Thus, the clinically unfavorable "TCL1 MBEN" subset revealed transcriptome signatures composed of cancer-associated signaling pathways and disclosed a high frequency of clinically relevant germline PTCH1/SUFU alterations. In contrast, gene expression profiles of tumors from the clinically favorable "TCL2 MBEN" subgroup were associated with activation of various neurometabolic and neurotransmission signaling pathways, and germline SHH-pathway gene mutations were extremely rare in this transcriptome cluster. "TCL2 MBEN" also revealed strong and ubiquitous expression of VSNL1 (visinin-like protein 1) both at the mRNA and protein level, which was correlated with a favorable clinical course. Thus, combining mutational and epigenetic profiling with transcriptome analysis including VSNL1 immunohistochemistry, MBEN patients could be stratified into clinical risk groups of potential value for subsequent treatment planning.
Subject(s)
Biomarkers, Tumor/metabolism , Cerebellar Neoplasms/genetics , Medulloblastoma/genetics , Neurocalcin/metabolism , Adolescent , Cerebellar Neoplasms/pathology , Child , Child, Preschool , Female , Gene Expression Profiling , Humans , Infant , Infant, Newborn , Male , Medulloblastoma/pathology , Prognosis , TranscriptomeABSTRACT
Epigenetic alterations, that is, disruption of DNA methylation and chromatin architecture, are now acknowledged as a universal feature of tumorigenesis. Medulloblastoma, a clinically challenging, malignant childhood brain tumour, is no exception. Despite much progress from recent genomics studies, with recurrent changes identified in each of the four distinct tumour subgroups (WNT-pathway-activated, SHH-pathway-activated, and the less-well-characterized Group 3 and Group 4), many cases still lack an obvious genetic driver. Here we present whole-genome bisulphite-sequencing data from thirty-four human and five murine tumours plus eight human and three murine normal controls, augmented with matched whole-genome, RNA and chromatin immunoprecipitation sequencing data. This comprehensive data set allowed us to decipher several features underlying the interplay between the genome, epigenome and transcriptome, and its effects on medulloblastoma pathophysiology. Most notable were highly prevalent regions of hypomethylation correlating with increased gene expression, extending tens of kilobases downstream of transcription start sites. Focal regions of low methylation linked to transcription-factor-binding sites shed light on differential transcriptional networks between subgroups, whereas increased methylation due to re-normalization of repressed chromatin in DNA methylation valleys was positively correlated with gene expression. Large, partially methylated domains affecting up to one-third of the genome showed increased mutation rates and gene silencing in a subgroup-specific fashion. Epigenetic alterations also affected novel medulloblastoma candidate genes (for example, LIN28B), resulting in alternative promoter usage and/or differential messenger RNA/microRNA expression. Analysis of mouse medulloblastoma and precursor-cell methylation demonstrated a somatic origin for many alterations. Our data provide insights into the epigenetic regulation of transcription and genome organization in medulloblastoma pathogenesis, which are probably also of importance in a wider developmental and disease context.
Subject(s)
DNA Methylation/genetics , Gene Expression Regulation, Neoplastic , Gene Silencing , Medulloblastoma/genetics , Sequence Analysis, DNA/methods , Animals , Binding Sites , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , Chromatin Immunoprecipitation , Female , Genome/genetics , Histones/metabolism , Humans , Medulloblastoma/pathology , Mice , Promoter Regions, Genetic/genetics , RNA-Binding Proteins/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.
Subject(s)
DNA-Binding Proteins/genetics , Enhancer Elements, Genetic/genetics , Genomic Structural Variation/genetics , Medulloblastoma/genetics , Oncogenes/genetics , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , Transcription Factors/genetics , Animals , Child , Chromosomes, Human, Pair 9/genetics , DNA-Binding Proteins/metabolism , Humans , Medulloblastoma/classification , Medulloblastoma/pathology , Mice , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolismABSTRACT
Desmoplastic/nodular medulloblastomas (DNMB) and medulloblastomas with extensive nodularity (MBEN) were outlined in the current WHO classification of tumors of the nervous system as two distinct histological MB variants. However, they are often considered as cognate SHH MB entities, and it is a reason why some clinical MB trials do not separate the patients with DNMB or MBEN histology. In the current study, we performed an integrated DNA/RNA-based molecular analysis of 83 DNMB and 36 MBEN to assess the etiopathogenetic relationship between these SHH MB variants. Methylation profiling revealed "infant" and "children" SHH MB clusters but neither DNMB nor MBEN composed separate epigenetic cohorts, and their profiles were intermixed within the "infant" cluster. In contrast, RNA-based transcriptional profiling disclosed that expression signatures of all MBEN were clustered separately from most of DNMB and a set of differentially expressed genes was identified. MBEN transcriptomes were enriched with genes associated with synaptic transmission, neuronal differentiation and metabolism, whereas DNMB profiling signatures included sets of genes involved in phototransduction and NOTCH signaling pathways. Thus, DNMB and MBEN are distinct tumor entities within the SHH MB family whose biology is determined by different transcriptional programs. Therefore, we recommend a transcriptome analysis as an optimal molecular tool to discriminate between DNMB and MBEN, which may be of benefit for patients' risk stratification in clinical trials. Molecular events identified in DNMB by RNA sequencing could be considered in the future as potent molecular targets for novel therapeutic interventions in treatment-resistant cases.
Subject(s)
Cerebellar Neoplasms/genetics , DNA Methylation , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Medulloblastoma/genetics , Transcription, Genetic , Adolescent , Age of Onset , Cerebellar Neoplasms/classification , Cerebellar Neoplasms/mortality , Cerebellar Neoplasms/pathology , Child , Child, Preschool , CpG Islands , DNA, Neoplasm/chemistry , Disease-Free Survival , Female , Gene Fusion , Hedgehog Proteins/physiology , High-Throughput Nucleotide Sequencing , Humans , Infant , Infant, Newborn , Kaplan-Meier Estimate , Male , Medulloblastoma/classification , Medulloblastoma/mortality , Medulloblastoma/pathology , Mutation , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplasm Recurrence, Local/genetics , Progression-Free Survival , Proportional Hazards Models , RNA, Messenger/biosynthesis , RNA, Neoplasm/genetics , Signal Transduction , TranscriptomeABSTRACT
Medulloblastoma with extensive nodularity (MBEN) is a rare histological variant of medulloblastoma (MB). These tumors are usually occurring in the first 3 years of life and are associated with good prognosis. Molecular analyses of MBEN, mostly limited to single cases or small series, have shown that they always classify as sonic hedgehog (SHH)-driven MB. Here, we have analyzed 25 MBEN through genome-wide DNA methylation, copy-number profiling and targeted next-generation sequencing. Results of these analyses were compared with molecular profiles of other SHH MB histological variants. As expected, the vast majority of MBEN (23/25) disclosed SHH-associated epigenetic signatures and mutational landscapes but, surprisingly, two MBEN were classified as Group 3/4 MB. Most MBEN classified as SHH MB displayed SHH-related and mutually exclusive mutations in either SUFU, or PTCH1, or SMO at similar frequencies. However, only SUFU mutations were also identified in the germ-line. Most of SUFU-associated MBEN eventually recurred but patients were treated successfully with second-line high-dose chemotherapy. Altogether, our data show that risk stratification even for well-recognizable histologies such as MBEN cannot rely on histology alone but should include additional molecular analyses such as methylation profiling and DNA sequencing. For all patients with "MBEN" histology, we recommend sequencing SUFU and PTCH1 in the tumor as well as in the germ-line for further clinical stratification and choice of the optimal treatment strategy upfront.
Subject(s)
Cerebellar Neoplasms/genetics , Hedgehog Proteins/genetics , Medulloblastoma/genetics , Repressor Proteins/genetics , Adolescent , Cancer Survivors , Cerebellar Neoplasms/pathology , Child , Child, Preschool , Cohort Studies , DNA Methylation/genetics , Female , Humans , Male , Medulloblastoma/pathology , Phosphopyruvate Hydratase/metabolism , Survival Analysis , Young AdultABSTRACT
According to the 2016 World Health Organization Classification of Tumors of the Central Nervous System (2016 CNS WHO), IDH-mutant astrocytic gliomas comprised WHO grade II diffuse astrocytoma, IDH-mutant (AIIIDHmut), WHO grade III anaplastic astrocytoma, IDH-mutant (AAIIIIDHmut), and WHO grade IV glioblastoma, IDH-mutant (GBMIDHmut). Notably, IDH gene status has been made the major criterion for classification while the manner of grading has remained unchanged: it is based on histological criteria that arose from studies which antedated knowledge of the importance of IDH status in diffuse astrocytic tumor prognostic assessment. Several studies have now demonstrated that the anticipated differences in survival between the newly defined AIIIDHmut and AAIIIIDHmut have lost their significance. In contrast, GBMIDHmut still exhibits a significantly worse outcome than its lower grade IDH-mutant counterparts. To address the problem of establishing prognostically significant grading for IDH-mutant astrocytic gliomas in the IDH era, we undertook a comprehensive study that included assessment of histological and genetic approaches to prognosis in these tumors. A discovery cohort of 211 IDH-mutant astrocytic gliomas with an extended observation was subjected to histological review, image analysis, and DNA methylation studies. Tumor group-specific methylation profiles and copy number variation (CNV) profiles were established for all gliomas. Algorithms for automated CNV analysis were developed. All tumors exhibiting 1p/19q codeletion were excluded from the series. We developed algorithms for grading, based on molecular, morphological and clinical data. Performance of these algorithms was compared with that of WHO grading. Three independent cohorts of 108, 154 and 224 IDH-mutant astrocytic gliomas were used to validate this approach. In the discovery cohort several molecular and clinical parameters were of prognostic relevance. Most relevant for overall survival (OS) was CDKN2A/B homozygous deletion. Other parameters with major influence were necrosis and the total number of CNV. Proliferation as assessed by mitotic count, which is a key parameter in 2016 CNS WHO grading, was of only minor influence. Employing the parameters most relevant for OS in our discovery set, we developed two models for grading these tumors. These models performed significantly better than WHO grading in both the discovery and the validation sets. Our novel algorithms for grading IDH-mutant astrocytic gliomas overcome the challenges caused by introduction of IDH status into the WHO classification of diffuse astrocytic tumors. We propose that these revised approaches be used for grading of these tumors and incorporated into future WHO criteria.
Subject(s)
Astrocytoma/genetics , Astrocytoma/pathology , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Isocitrate Dehydrogenase/genetics , Mutation/genetics , Adolescent , Adult , Aged , Algorithms , Astrocytoma/mortality , Brain Neoplasms/mortality , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Ki-67 Antigen/metabolism , Male , Middle Aged , Models, Biological , Neoplasm Grading , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , World Health Organization , Young AdultABSTRACT
Posterior fossa ependymoma comprise three distinct molecular variants, termed PF-EPN-A (PFA), PF-EPN-B (PFB), and PF-EPN-SE (subependymoma). Clinically, they are very disparate and PFB tumors are currently being considered for a trial of radiation avoidance. However, to move forward, unraveling the heterogeneity within PFB would be highly desirable. To discern the molecular heterogeneity within PFB, we performed an integrated analysis consisting of DNA methylation profiling, copy-number profiling, gene expression profiling, and clinical correlation across a cohort of 212 primary posterior fossa PFB tumors. Unsupervised spectral clustering and t-SNE analysis of genome-wide methylation data revealed five distinct subtypes of PFB tumors, termed PFB1-5, with distinct demographics, copy-number alterations, and gene expression profiles. All PFB subtypes were distinct from PFA and posterior fossa subependymomas. Of the five subtypes, PFB4 and PFB5 are more discrete, consisting of younger and older patients, respectively, with a strong female-gender enrichment in PFB5 (age: p = 0.011, gender: p = 0.04). Broad copy-number aberrations were common; however, many events such as chromosome 2 loss, 5 gain, and 17 loss were enriched in specific subtypes and 1q gain was enriched in PFB1. Late relapses were common across all five subtypes, but deaths were uncommon and present in only two subtypes (PFB1 and PFB3). Unlike the case in PFA ependymoma, 1q gain was not a robust marker of poor progression-free survival; however, chromosome 13q loss may represent a novel marker for risk stratification across the spectrum of PFB subtypes. Similar to PFA ependymoma, there exists a significant intertumoral heterogeneity within PFB, with distinct molecular subtypes identified. Even when accounting for this heterogeneity, extent of resection remains the strongest predictor of poor outcome. However, this biological heterogeneity must be accounted for in future preclinical modeling and personalized therapies.
Subject(s)
DNA Copy Number Variations/genetics , Ependymoma/classification , Ependymoma/genetics , Infratentorial Neoplasms/classification , Infratentorial Neoplasms/genetics , Adolescent , Adult , Age Factors , Child , Cohort Studies , DNA Methylation/genetics , Ependymoma/pathology , Ependymoma/surgery , Female , Gene Expression Profiling , Humans , Infratentorial Neoplasms/pathology , Infratentorial Neoplasms/surgery , Kaplan-Meier Estimate , Male , Microarray Analysis , Middle Aged , Young AdultABSTRACT
Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromatin/genetics , Glioblastoma/genetics , Histones/genetics , Mutation/genetics , Adaptor Proteins, Signal Transducing/genetics , Base Sequence , Child , Chromatin/metabolism , Co-Repressor Proteins , DNA Helicases/genetics , DNA Mutational Analysis , Exome/genetics , Gene Expression Profiling , Histones/metabolism , Humans , Molecular Chaperones , Molecular Sequence Data , Nuclear Proteins/genetics , Telomere/genetics , Tumor Suppressor Protein p53/genetics , X-linked Nuclear ProteinABSTRACT
Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.
Subject(s)
Cerebellar Neoplasms/genetics , Genome, Human/genetics , Medulloblastoma/genetics , Aging/genetics , Amino Acid Sequence , Cell Transformation, Neoplastic , Cerebellar Neoplasms/classification , Cerebellar Neoplasms/diagnosis , Cerebellar Neoplasms/pathology , Child , Chromatin/metabolism , Chromosomes, Human/genetics , DEAD-box RNA Helicases/genetics , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Genomics , Hedgehog Proteins/metabolism , High-Throughput Nucleotide Sequencing , Histone Demethylases/genetics , Humans , Medulloblastoma/classification , Medulloblastoma/diagnosis , Medulloblastoma/pathology , Methylation , Mutation/genetics , Mutation Rate , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Oncogene Proteins, Fusion/genetics , Patched Receptors , Patched-1 Receptor , Phosphoprotein Phosphatases/genetics , Polyploidy , Receptors, Cell Surface/genetics , Sequence Analysis, RNA , Signal Transduction , T-Box Domain Proteins/genetics , Transcription Factors/genetics , Wnt Proteins/metabolism , beta Catenin/geneticsABSTRACT
Pediatric glioblastoma (pedGBM) is an extremely aggressive pediatric brain tumor, accounting for ~6% of all central nervous system neoplasms in children. Approximately half of pedGBM harbor recurrent somatic mutations in histone 3 variants or, infrequently, IDH1/2. The remaining subset of pedGBM is highly heterogeneous, and displays a variety of genomic and epigenetic features. In the current study, we aimed to further stratify an H3-/IDH-wild type (wt) pedGBM cohort assessed through genome-wide molecular profiling. As a result, we identified three molecular subtypes of these tumors, differing in their genomic and epigenetic signatures as well as in their clinical behavior. We designated these subtypes 'pedGBM_MYCN' (enriched for MYCN amplification), 'pedGBM_RTK1' (enriched for PDGFRA amplification) and 'pedGBM_RTK2' (enriched for EGFR amplification). These molecular subtypes were associated with significantly different outcomes, i.e. pedGBM_RTK2 tumors show a significantly longer survival time (median OS 44 months), pedGBM_MYCN display extremely poor outcomes (median OS 14 months), and pedGBM_RTK1 tumors harbor an intermediate prognosis. In addition, the various molecular subtypes of H3-/IDH-wt pedGBM were clearly distinguishable from their adult counterparts, underlining their biological distinctiveness. In conclusion, our study demonstrates significant molecular heterogeneity of H3-/IDH-wt pedGBM in terms of DNA methylation and cytogenetic alterations. The recognition of three molecular subtypes of H3-/IDH-wt pedGBM further revealed close correlations with biological parameters and clinical outcomes and may therefore, be predictive of response to standard treatment protocols, but could also be useful for stratification for novel, molecularly based therapies.
Subject(s)
Brain Neoplasms/genetics , Glioblastoma/genetics , Histones/genetics , Isocitrate Dehydrogenase/genetics , Mutation , Adolescent , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Carcinogenesis/genetics , Child , Child, Preschool , DNA Methylation , DNA Mutational Analysis , Female , Glioblastoma/metabolism , Glioblastoma/pathology , Histones/metabolism , Humans , Isocitrate Dehydrogenase/metabolism , Male , Prognosis , Promoter Regions, GeneticABSTRACT
Intraocular medulloepithelioma (IO-MEPL) is an uncommon embryonal neuroepithelial neoplasm of the eye. Little is known about the cytogenetics, molecular biology, and pathogenesis of this tumor. In the present study we investigated the mutational landscape of 19 IO-MEPL using targeted next-generation sequencing. Routinely prepared paraffin-embedded samples were assessed with high-coverage genome sequencing on the Illumina NextSeq 500 platform using a customized gene panel set covering the coding region of 130 genes. This revealed several notable genomic alterations, including mutations of DICER1 (6 tumors) and KMT2D (also known as MLL2; 5 tumors)-which are frequently recurrent and mutually exclusive molecular events for IO-MEPL. Non-recurrent mutations in the cancer-associated genes BRCA2, BRCA1, NOTCH2, CDH1, and GSE1 were also identified. IO-MEPL samples harboring a DICER1 mutation disclosed few chromosomal alterations and formed a separate DNA methylation cluster, indicating potential differences in genetic and epigenetic events arising perhaps from the presence of this aberration in the tumor genome. The high proportion of recurrent somatic DICER1 and KMT2D mutations in this series of sporadic IO-MEPL points to their likely important roles in the molecular pathogenesis of these rare embryonal tumors, and perhaps suggests the existence of distinct molecular variants of IO-MEPL. Although the precise role of these recurrent mutations in the development of IO-MEPL, and their relationship to pro-oncogenic molecular mechanisms, have yet to be determined, unraveling their roles could eventually be exploited for nonsurgical therapies of these neoplasms.
Subject(s)
DEAD-box RNA Helicases/genetics , DNA-Binding Proteins/genetics , Eye Neoplasms/genetics , Mutation , Neoplasm Proteins/genetics , Neuroectodermal Tumors, Primitive/genetics , Ribonuclease III/genetics , Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Male , Middle AgedABSTRACT
In contrast to the relative morphological uniformity of histone H3 K27-mutant high-grade gliomas, H3 G34-mutant tumors present as a histopathologically heterogeneous group of neoplasms, with microscopic characteristics typical of either glioblastoma (GBM) or central nervous system primitive neuroectodermal tumors (CNS-PNET). In the current study, we performed an integrative clinical, histopathological and molecular analysis of 81 G34-mutant CNS tumors. Routinely prepared tumor tissues were investigated for genomic and epigenomic alterations. Despite their divergent histopathological appearance, CNS tumors with H3.3 G34 mutations displayed uniform epigenetic signatures, suggesting a single biological origin. Comparative cytogenetic analysis with other GBM subtypes disclosed a high frequency and high specificity of 3q and 4q loss across G34-mutant tumors. PDGFRA amplification was more common in cases with GBM than with PNET morphology (36 vs. 5 %, respectively), while CCND2 amplifications showed the opposite trend (5 vs. 27 %). Survival analysis revealed the presence of amplified oncogene(s) and MGMT methylation as independent prognostic markers for poor and favorable outcomes, respectively. No difference in outcome was found between morphological variants (GBM vs. PNET). Thus, different histological variants of G34-mutant CNS tumors likely comprise a single biological entity (high-grade glioma with H3 G34 mutation, HGG_G34), which should be outlined in future diagnostic and therapeutic classifications. Screening for H3.3 G34 mutation should therefore be recommended as a routine diagnostic marker for supratentorial CNS tumors across a broad histological spectrum.