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.

2nd Department of Pediatrics Semmelweis University Budapest Hungary

Aix Marseille University Neurophysiopathology Institute CNRS Marseille France

Ann and Robert H Lurie Children's Hospital of Chicago Chicago IL USA

Brain Tumor Program Texas Children's Cancer Center Department of Pediatrics Baylor College of Medicine Houston TX USA

Center for CNS Tumors Comprehensive Cancer Center Tübingen Stuttgart University Hospital of Tübingen Tübingen Germany

Clinical Cooperation Unit Neuropathology German Cancer Research Center Heidelberg Germany

Clinical Cooperation Unit Pediatric Oncology German Cancer Research Center Heidelberg Germany

Department for Pediatric Hematology and Oncology University Hospital Hamburg Eppendorf Hamburg Germany

Department of Cell Systems and Anatomy University of Texas Health at San Antonio San Antonio TX USA

Department of Clinical Sciences Lund Section of Neurosurgery Faculty of Medicine Lund University Lund Sweden

Department of Neuropathology Heidelberg University Hospital Heidelberg Germany

Department of Neuropathology Institute of Pathology and Neuropathology University Hospital of Tübingen Tübingen Germany

Department of Neuropathology NN Burdenko Neurosurgical Institute Moscow Russia

Department of Oncogenomics Academic Medical Center Amsterdam The Netherlands

Department of Pathology Amsterdam University Medical Centers location VUmc Amsterdam The Netherlands

Department of Pathology and Laboratory Medicine University of Calgary Calgary Alberta Canada

Department of Pathology APHM Marseille France

Department of Pathology Boston Children's Hospital Harvard Medical School Boston MA USA

Department of Pathology Children's Memorial Health Institute Warsaw Poland

Department of Pathology Montpellier University Medical Center Montpellier France

Department of Pathology NYU Langone Health New York NY USA

Department of Pathology St Jude Children's Research Hospital Memphis TN USA

Department of Pathology Toulouse University Hospital Toulouse France

Department of Pediatric Hematology and Oncology University Hospital Motol Prague Czech Republic

Department of Pediatric Hematology and Oncology University Medical Center Hamburg Eppendorf Hamburg Germany

Department of Pediatric Oncology Hematology and Clinical Immunology Medical Faculty University Hospital Düsseldorf Düsseldorf Germany

Department of Pediatric Oncology Hematology and Immunology Heidelberg University Hospital Heidelberg Germany

Department of Pediatric Oncology Hematology Charité University Medicine Berlin Germany

Department of Pediatrics McGill University Health Center McGill University Montreal Quebec Canada

Department of Pediatrics Medical Biophysics Laboratory Medicine and Pathobiology University of Toronto Toronto Ontario Canada

Department of Pediatrics Pediatric Oncology and Hematology Faculty of Medicine and University Hospital Cologne University of Cologne Cologne Germany

Department of Radiological Oncological and Anatomopathological Sciences Sapienza University of Rome Rome Italy

Division of Hematology Oncology Arthur and Sonia Labatt Brain Tumour Research Centre The Hospital for Sick Children Toronto Ontario Canada

Division of Molecular Genetics German Cancer Research Center Heidelberg Germany

Division of Neurosurgery Arthur and Sonia Labatt Brain Tumor Research Center Hospital for Sick Children University of Toronto Toronto Ontario Canada

Division of Pediatric Hematology Oncology NYU Langone Medical Center The Stephen D Hassenfeld Children's Center for Cancer and Blood Disorders New York NY USA

Division of Pediatric Neurooncology German Cancer Research Center Heidelberg Germany

European Molecular Biology Laboratory Genome Biology Unit Heidelberg Germany

Genomics and Proteomics Core Facility High Throughput Sequencing Unit German Cancer Research Center Heidelberg Germany

German Cancer Consortium Heidelberg Germany

Greehey Children's Cancer Research Institute University of Texas Health at San Antonio San Antonio TX USA

Hopp Children's Cancer Center Heidelberg Germany

INSERM U1037 Cancer Research Center of Toulouse Toulouse France

INSERM U830 Laboratory of Translational Research in Pediatric Oncology SIREDO Pediatric Oncology Center Paris Sciences Lettres Research University Curie Institute Paris France

Institute for Neuroscience of Montpellier INSERM U1051 Montpellier University Hospital Montpellier France

Institute of Neurology Medical University of Vienna Vienna Austria

Institute of Neuropathology Brain Tumor Reference Center of the German Society of Neuropathology and Neuroanatomy University of Bonn Medical Center Bonn Germany

Institute of Neuropathology University Hospital Münster Münster Germany

Institute of Neuropathology University Medical Center Hamburg Eppendorf Hamburg Germany

IRCCS Neuromed Mediterranean Neurological Institute Pozzilli Italy

Mays Cancer Center University of Texas Health at San Antonio San Antonio TX USA

Northwestern University Feinberg School of Medicine Chicago IL USA

Omics IT and Data Management Core Facility German Cancer Research Center Heidelberg Germany

Paris Sciences et Lettres Research University Institut Curie Hospital Laboratory of Somatic Genetics Paris France

Pediatric Glioma Research Group German Cancer Research Center Heidelberg Germany

Pediatric Hematology and Oncology Division University Hospital São João Alameda Hernani Monteiro Porto Portugal

Pediatric Oncology Department SIREDO Pediatric Oncology Centre Curie Institute Paris France

Princess Máxima Center for Pediatric Oncology Utrecht The Netherlands

Research Institute Children's Cancer Center Hamburg Germany

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