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There is a growing role for mutations affecting histone linker and histone core-encoding genes across several adult and pediatric cancers. However, the extent to which somatic histone mutations may bridge across different cancers as common tumorigenic events – particularly in the context of pediatric CNS tumors – remains unclear. To address this knowledge gap, we set out to define a comprehensive pan-cancer landscape of somatic histone mutations. We first queried the ICGC PCAWG and TCGA Pan-Cancer Atlas representing >12,500 adult and pediatric cancer patients. We found lymphomas to be most enriched for histone mutations (50–75%) and, in particular, for mutations in linker histones (HIST1H1B-E), yet also in specific core histone genes (eg, HIST2H2BE). Moreover, we observed a significant enrichment of histone mutations in adult high-grade vs low-grade gliomas (10% vs 6%, P

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.

We compared 24 primary pediatric T‐cell acute lymphoblastic leukemias (T‐ALL) collected at the time of initial diagnosis and relapse from 12 patients and 24 matched patient‐derived xenografts (PDXs). DNA methylation profile was preserved in PDX mice in 97.5% of the promoters (ρ = 0.99). Similarly, the genome‐wide chromatin accessibility (ATAC‐Seq) was preserved remarkably well (ρ = 0.96). Interestingly, both the ATAC regions, which showed a significant decrease in accessibility in PDXs and the regions hypermethylated in PDXs, were associated with immune response, which might reflect the immune deficiency of the mice and potentially the incomplete interaction between murine cytokines and human receptors. The longitudinal approach of this study allowed an observation that samples collected from patients who developed a type 1 relapse (clonal mutations maintained at relapse) preserved their genomic composition; whereas in patients who developed a type 2 relapse (subset of clonal mutations lost at relapse), the preservation of the leukemia's composition was more variable. In sum, this study underlines the remarkable genomic stability, and for the first time documents the preservation of the epigenomic landscape in T‐ALL‐derived PDX models.

INTRODUCTION DMG-ACT (DMG- multi-arm Adaptive and Combinatorial Trial) aims to implement a highly innovative clinical trial design of combinatorial arms for patients with diffuse midline gliomas (DMGs) at all disease stages that is adaptive to pre-clinical data generated in eight collaborating institutions. The goals of the team are to: i) rapidly identify and validate promising drugs for clinical use, and ii) predict biomarkers for promising drugs. METHODS In vitro (n=15) and in vivo (n=8) models of DMGs across seven institutions were used to assess single and combination treatments with ONC201, ONC206, marizomib, panobinostat, Val-083, and TAK228. In vivo pharmacokinetic assays using clinically relevant dosing of ONC201, ONC206, and panobinostat were performed. Predictive biomarkers for ONC201 and ONC206 were identified using extensive molecular assays including CRISPR, RNAseq, ELISA, FACS, and IHC. RESULTS Inhibitory concentrations (IC50) were established and validated across participating sites. In vivo validation of single and combination drug assays confirmed drug efficacy as increased survival for: ONC201 (p=0.01), ONC206 (p=0.01), ONC201+ONC206 (p=0.02), and ONC201+panobinostat (p=0.01). Marizomib showed toxicity in murine/zebrafish PDXs models. Murine pharmacokinetic analysis showed peak brain levels of ONC201 and ONC206 above pre-clinical IC50. Molecular testing and analyses of existing drug screen across 537 cancer cell lines validated mitochondrial stress and ATF4 as the main targets induced by ONC201/6. CONCLUSION Thorough preclinical testing in a multi-site laboratory setting is feasible and identified ONC201 in combination with ONC206 as promising therapeutics for DMGs. Preclinical and correlative-clinical studies are ongoing.

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.

Introduction: Embryonal tumors with multilayered rosettes (ETMRs) are aggressive brain tumors that occur mainly in infants. Patients face a very poor prognosis with a median overall survival of ~12 months after diagnosis. The tumors harbor in ~90% of all cases amplification of a miRNA cluster on chromosome 19 (C19MC) that is thought to be the driver of the disease. However, current treatment options are lacking as (a) the mechanisms downstream of C19MC are poorly understood and (b) the drivers in cases lacking the C19MC aberration are unknown. To develop better treatment protocols for ETMR patients, more insight is needed in what is driving these tumors and how that can be targeted. Materials and Methods: To investigate the genomic and epigenomic landscape of ETMR in depth, we collected 193 ETMR samples and 23 matched relapses and performed DNA methylation profiling on all and DNA (whole genome, whole exome, and panel) sequencing and mRNA and miRNA transcriptome analysis on a subset of them. The BT183 ETMR cell line was used for drug treatments. Results: Among the 22 tumors without C19MC amplification, we identified 8 cases with truncating DICER1 germline mutations in one allele and somatic missense mutations in the RNASE III domain in the other allele. No DICER1 mutations were identified in C19MC amplified cases. In addition, structural variations (SVs) affecting C19MC were found in 3 other C19MC nonamplified cases and amplification of another miRNA cluster, miR-17-92, in 2 other cases. However, despite the presence of different genetic aberrations, based on DNA methylation and transcriptome profiling no molecular subgrouping was observed within our cohort. Whole-genome sequencing revealed an overall low recurrence and conservation of SNVs but strong conservation of SVs from primary tumors to relapses, especially surrounding C19MC. Moreover, many newly acquired SNVs in the relapses are associated to a new cisplatin treatment-related mutational signature. SVs detected in ETMRs significantly colocalized with R-loops, structures that form upon a collision of replication and transcription and are associated to increased levels of chromosomal instability, which is frequently observed in ETMRs. Using a DICER1 KO model, we found that global deregulation of miRNAs led to increased levels of R-loops and R-loop associated chromosomal instability. Finally, we show that a combination of topoisomerase and PARP inhibitors is highly synergistic and strongly increased the levels of both R-loops and DNA damage in ETMR cells and effectively killed the cells. Conclusions: Our results show that genomically instable ETMR cells are vulnerable to further increases in chromosomal instability, knowledge that may lead to new treatment strategies for ETMR patients and possibly other cancers with high levels of R-loops. Citation Format: Sander Lambo, Susanne Grübner, Tobias Rausch, Sebastian Waszak, Christin Schmidt, Sonja Krausert, Loreen Weichert, Aparna Gorthi, Carolina Romero, Annie Huang, Julia Schueler, Jan Korbel, Alexander Bishop, Stefan Pfister, Andrey Korshunov, Marcel Kool. Molecular characterization of ETMRs reveals role for R-loop mediated genomic instability and new treatment options [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A39.

Embryonal Tumors with Multilayered Rosettes (ETMRs) are aggressive pediatric brain tumors mainly occurring in infants. In order to develop alternative treatment strategies for this deadly disease there is an urgent need for better understanding the mechanisms driving these tumors. We therefore analyzed a cohort of 60 ETMRs using whole genome and panel sequencing, DNA methylation, mRNA expression profiling, and miRNA sequencing. The genetic hallmark of ETMRs is amplification of miRNA cluster C19MC fused to TTYH1 present in ~90% of all ETMRs. ETMRs lacking the C19MC amplification are biologically highly similar to tumors with C19MC amplification, indicating that they do not represent a distinct subgroup. DNA sequencing revealed germline mutations affecting DNA repair genes or miRNA processing genes in a subset of cases, while tumor specific mutations included genes involved in the TP53-, SHH- WNT-, or miRNA processing pathways. Prevalence of mutations in miRNA processing pathways are specifically high in tumors without C19MC amplification, however overall recurrence of mutations is low within our cohort. We also detect high recurrence of genomic instability shown by pluriploidy, large abundance of CNVs, highly upregulated DNA repair pathways and chromothripsis primarily centered around the C19MC amplicon. Structural variations throughout the genome, including the C19MC amplicon, are highly conserved from primary tumor to relapse while other mutations show low conservation. These results suggest ETMR is an entity driven by structural variations rather than single nucleotide variants and could lead to new treatment options, specifically targeting genomic instability, to be tested in preclinical model systems of ETMR.

BACKGROUND: iMB has inferior survival to older children principally due to radiation-sparing therapy. To better understand which patients may benefit from radiation-sparing protocols, we describe the molecular landscape of iMB and report the iMB outcome on the SJYC07 trial designed to defer, reduce, or delay radiation exposure. METHODS: We assembled a molecular cohort of 190 iMBs and a SJYC07 trial cohort of 81 iMBs. Tumors were sub-classified into molecular subgroups based on DNA methylation profiles and overlaid with mutations and copy-number alterations. PFS and OS for the SJYC07 cohort was estimated across clinical risk groups, consensus molecular subgroups, and in the context of novel MB subtypes. RESULTS: Computational analysis of DNA methylation array data divided iMB into three of the four consensus subgroups: SHH, G3, and G4 (absent WNT). Clinical outcome of iMB(SHH) was superior to iMB(Group3/Group4) (5-year PFS: 51 ± 8% vs 11 ± 10%, P

In this Article, author Benedikt Brors was erroneously associated with affiliation number '8' (Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee, USA); the author's two other affiliations (affiliations '3' and '7', both at the German Cancer Research Center (DKFZ)) were correct. This has been corrected online.

Background: Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines. Methods: In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGroup3), and group 4 (MBGroup4). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma. Findings: We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MBSHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MBSHH subgroup). Patients with germline APC mutations developed MBWNT and accounted for most (five [71%] of seven) cases of MBWNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MBSHH. Germline TP53 mutations presented only in childhood patients in the MBSHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MBSHH, MBGroup3, and MBGroup4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40–69) and 5-year overall survival was 65% (95% CI 52–81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes. Interpretation: Genetic counselling and testing should be used as a standard-of-care procedure in patients with MBWNT and MBSHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and molecular tumour characteristics. Funding: German Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario.

SummaryChromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

We present Butler, a computational framework developed in the context of the international Pan-cancer Analysis of Whole Genomes (PCAWG)1 project to overcome the challenges of orchestrating analyses of thousands of human genomes on the cloud. Butler operates equally well on public and academic clouds. This highly flexible framework facilitates management of virtual cloud infrastructure, software configuration, genomics workflow development, and provides unique capabilities in workflow execution management. By comprehensively collecting and analysing metrics and logs, performing anomaly detection as well as notification and cluster self-healing, Butler enables large-scale analytical processing of human genomes with 43% increased throughput compared to prior setups. Butler was key for delivering the germline genetic variant call-sets in 2,834 cancer genomes analysed by PCAWG1.

We present the most comprehensive catalogue of cancer-associated gene alterations through characterization of tumor transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes project. Using matched whole-genome sequencing data, we attributed RNA alterations to germline and somatic DNA alterations, revealing likely genetic mechanisms. We identified 444 associations of gene expression with somatic non-coding single-nucleotide variants. We found 1,872 splicing alterations associated with somatic mutation in intronic regions, including novel exonization events associated with Alu elements. Somatic copy number alterations were the major driver of total gene and allele-specific expression (ASE) variation. Additionally, 82% of gene fusions had structural variant support, including 75 of a novel class called “bridged” fusions, in which a third genomic location bridged two different genes. Globally, we observe transcriptomic alteration signatures that differ between cancer types and have associations with DNA mutational signatures. Given this unique dataset of RNA alterations, we also identified 1,012 genes significantly altered through both DNA and RNA mechanisms. Our study represents an extensive catalog of RNA alterations and reveals new insights into the heterogeneous molecular mechanisms of cancer gene alterations.

About half of all cancers have somatic integrations of retrotransposons. To characterize their role in oncogenesis, we analyzed the patterns and mechanisms of somatic retrotransposition in 2,954 cancer genomes from 37 histological cancer subtypes. We identified 19,166 somatically acquired retrotransposition events, affecting 35% of samples, and spanning a range of event types. L1 insertions emerged as the first most frequent type of somatic structural variation in esophageal adenocarcinoma, and the second most frequent in head-and-neck and colorectal cancers. Aberrant L1 integrations can delete megabase-scale regions of a chromosome, sometimes removing tumour suppressor genes, as well as inducing complex translocations and large-scale duplications. Somatic retrotranspositions can also initiate breakage-fusion-bridge cycles, leading to high-level amplification of oncogenes. These observations illuminate a relevant role of L1 retrotransposition in remodeling the cancer genome, with potential implications in the development of human tumours.

Motivation: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. Results: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent–daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. Availability: The R package absfilter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter Contact: sebastian.waszak@epfl.ch or bart.deplancke@epfl.ch Supplementary information: Supplementary data are available at Bioinformatics online.

Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.

ETMRs are aggressive embryonal brain tumors with a very poor prognosis, having a median overall survival of ~12 months after diagnosis. ETMRs harbour in ~90% of all cases amplification of a miRNA cluster on chromosome 19 (C19MC) that is thought to be the driver of the disease. However, current treatment options are lacking as (a) the mechanisms downstream of C19MC are poorly understood and (b) the drivers in cases lacking the C19MC aberration are unknown. To investigate the genomic landscape of ETMR, we collected 186 ETMR samples and 23 matched relapses. Interestingly, among the 18 tumors without C19MC amplification, we identified five cases with truncating DICER1 germline mutations in one allele and somatic missense mutations in the RNASE III domain in the other allele. In addition, SVs affecting C19MC were found in three other cases and amplification of the miR-17–92 cluster in another two cases. However, despite the presence of different genetic aberrations, based on DNA methylation profiling no molecular subgrouping was observed within our cohort. Whole-genome sequencing revealed an overall low recurrence and conservation of SNVs but strong conservation of SVs from primary tumors to relapses especially surrounding C19MC. SVs detected in ETMRs significantly co-localized with R-loops, structures that form upon a collision of replication and transcription and are associated to increased levels of chromosomal instability, which is frequently observed in ETMRs. Using a DICER1 KO model we found that global deregulation of miRNAs led to increased levels of R-loops and R-loop associated chromosomal instability. Finally, we show that treating ETMR cells with topoisomerase and PARP inhibitors strongly increased the levels of both R-loops and DNA damage and effectively killed the cells. Our results show that genomically instable ETMR cells are vulnerable to further increases in chromosomal instability, knowledge that may lead to new treatment strategies in the future.

DNA Differences The extent to which genetic variation affects an individual's phenotype has been difficult to predict because the majority of variation lies outside the coding regions of genes. Now, three studies examine the extent to which genetic variation affects the chromatin of individuals with diverse ancestry and genetic variation (see the Perspective by Furey and Sethupathy ). Kasowski et al. (p. 750 , published online 17 October) examined how genetic variation affects differences in chromatin states and their correlation to histone modifications, as well as more general DNA binding factors. Kilpinen et al. (p. 744 , published online 17 October) document how genetic variation is linked to allelic specificity in transcription factor binding, histone modifications, and transcription. McVicker et al. (p. 747 , published online 17 October) identified how quantitative trait loci affect histone modifications in Yoruban individuals and established which specific transcription factors affect such modifications.

Summary Identifying the earliest somatic changes in prostate cancer can give important insights into tumor evolution and aids in stratifying high- from low-risk disease. We integrated whole genome, transcriptome and methylome analysis of early-onset prostate cancers (diagnosis ???55 years). Characterization across 292 prostate cancer genomes revealed age-related genomic alterations and a clock-like enzymatic-driven mutational process contributing to the earliest mutations in prostate cancer patients. Our integrative analysis identified four molecular subgroups, including a particularly aggressive subgroup with recurrent duplications associated with increased expression of ESRP1, which we validate in 12,000 tissue microarray tumors. Finally, we combined the patterns of molecular co-occurrence and risk-based subgroup information to deconvolve the molecular and clinical trajectories of prostate cancer from single patient samples.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is classified according to surface marker expression. In order to reveal the cells of origin in pediatric T-ALL and to understand mechanisms of relapse we used ATAC-Seq (Assay for Transposase-Accessible Chromatin Sequencing) to compare chromatin accessibility landscapes of healthy T-cell precursors to those of T-ALL cells obtained at initial diagnosis (INI) and relapse (REL). We have FACS sorted 7 differentiation stages of normal T-cell precursors contained in the thymus of infants undergoing cardiac surgery (DN2, DN3, ISP, DPCD3-, DPCD3+, CD4+ and CD8+) and subjected these to ATAC-Seq. Unsupervised learning by principal component analysis (PCA) clustered sorted populations according to the maturation stage, demonstrating that regulatory chromatin signatures of thymocytes are highly stage-specific and re-shaped during T-cell differentiation. We next compared normal T-cell precursors at different stages of maturation to pediatric T-ALLs and found fundamental differences with 30% of open chromatin regions to be more and 28% being less accessible in T-ALL (DESeq2, padj We then subjected the ATAC-seq data of all matched leukemia samples obtained at initial disease and at relapse to PCA. INI and REL samples derived from the same patient always clustered in close proximity and were separated according to the T-ALL driving fusion genes. A global analysis of differential accessibility revealed only 0.26% of ATAC-regions to be less- or more-accessible at relapse when compared to the matched initial samples (DESeq2, padj Moreover, we trained the deconvolution algorithm CIBERSORT to recognize particular T-cell differentiation stages using ATAC-profiles of the 7 FACS-sorted healthy T-cell populations. We used regulatory chromatin landscape of non-sorted (total) thymus to assess the accuracy of deconvolution. Comparison of predicted fractions in total thymus to FACS measurements revealed highly accurate identification of the maturation stages (r2 = 0.95). CIBERSORT analysis confirmed that the profiles were largely preserved between INI and REL of each sample pair. Notably, however, while in T-ALLs that later developed into a type 1 relapse only one type of early T-cell progenitor dominated the deconvolution profile, T-ALLs that developed into a type 2 relapse showed heterogeneous profiles with contributions of progenitors at different maturation stages. In sum, these epigenomic analyses revealed that the chromatin landscape of normal T-cell precursors evolves in the course of thymic maturation and that the early maturation stages are the likely origin of T-ALL cells. Remarkably, pediatric T-ALLs that later develop a type 2 relapse consist of subclones with a variable profile of chromatin accessibility that define different stages of maturation. These data indicate that T-ALLs with the propensity to develop a type 2 relapse differ from type 1 in that they originate from early precursors that carry the potential of further development into different stages of maturation before the leukemia becomes apparent with a highly subclonal pattern. Disclosures Muckenthaler: Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals., Author Summary One of the principal challenges in current biology is to understand the relationship between genetic and phenotypic variation. The increasing availability of genomic variation maps of human as well as of model organism populations (mouse and Arabidopsis) constitutes an important step towards meeting this challenge. However, despite its excellent track record as a premier model to understand genome function, no genome-wide variation data beyond single-nucleotide variants and microsatellites are currently available for D. melanogaster. Here, we present a comprehensive, nucleotide-resolution catalogue of variants of various types (single-nucleotide, multi-nucleotide, and structural variants) for 39 wild-derived inbred D. melanogaster lines based on high-throughput sequencing. This catalogue confirms that non–SNP variants account for more than half of genomic variation, allowing us to provide new insights into the non-random distribution of variants in the Drosophila genome. We further present genome-wide cis-associations with gene expression based on whole adult fly microarray data, revealing significant associations for about 2,000 genes. Most associations are sex-specific, providing evidence for a decoupling of the genomic, regulatory architecture between males and females.

Copy-number variants (CNVs) form an abundant class of genetic variation with a presumed widespread impact on individual traits. While recent advances, such as the population-scale sequencing of human genomes, facilitated the fine-scale mapping of CNVs, the phenotypic impact of most of these CNVs remains unclear. By relating copy-number genotypes to transcriptome sequencing data, we have evaluated the impact of CNVs, mapped at fine scale, on gene expression. Based on data from 129 individuals with ancestry from two populations, we identified CNVs associated with the expression of 110 genes, with 13% of the associations involving complex, multiallelic CNVs. Categorization of CNVs according to variant type, size, and gene overlap enabled us to examine the impact of different CNV classes on expression variation. While many small (

Embryonal Tumors with Multilayered Rosettes (ETMRs) are pediatric brain tumors mainly occurring in infants. Characteristic to ETMRs is the highly recurrent (~90%) amplification of the C19MC miRNA cluster fused to TTYH1 that drives the expression of this cluster. As the overall survival of these patients is very poor, there is an urgent need for a better understanding of these tumors that may lead to other treatment strategies. Whole genome and panel sequencing data have been generated for 60 ETMRs and matching germline when available. Data have been complemented with DNA methylation profiling and m(i)RNA sequencing data. Our results show that ETMR is a single disease entity without molecular subgroups. ETMRs lacking the C19MC amplification (~10%) are highly similar to tumors with C19MC amplification, based on methylation and m(i)RNA profiling, indicating that they do not represent a distinct subgroup. Germline sequencing revealed mutations in genes involved in DNA repair or miRNA processing, while tumor specific mutations included genes involved in the TP53-, SHH-, WNT-, or miRNA processing pathways. These pathways are also highly upregulated compared to other pediatric brain tumors. Mutations in DNA repair, miRNA processing, structural variations (SVs) and mutations in close proximity of SVs occur at high allele frequencies and are conserved in recurrent tumors while many other SNVs are lost. These data suggest that C19MC amplification/fusion, miRNA processing and DNA repair defects are the early (driving) events in tumor formation while aberrations involving for instance the SHH and WNT pathways are later (passenger) events. Aside from frequent and recurrent copy number changes, ETMRs show pluriploidy, complex rearrangements and strong presence of R-loops suggesting that ETMR genomes are highly unstable. We identified a high number of R-loops in the region forming the C19MC aberration and an enrichment of breakpoints in other R-loop forming regions. This may suggest a role for R-loops in both tumor progression and initiation. Finally, we tested whether further inducing the number of R-loops in these tumors may increase replication stress and cell death. Indeed, topoisomerase inhibition coupled to PARP inhibition increased the amount of R-loops and acted synergistically in killing ETMR cells. These data show that targeting the genomic instability in ETMRs could be a viable treatment option for treating ETMR patients. Citation Format: Sander Lambo, Andrey Korshunov, Christin Schmidt, Carolina Romero, Aparna Gorthi, Sonja Krausert, Tobias Rausch, Susanne Gröbner, Sebastian Brabetz, Sebastian Waszak, Alexander J. Bishop, Stefan Pfister, Marcel Kool. Targeting genomic instability in embryonal tumors with multilayered rosettes (ETMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3172.