Hétérogénéïté Génétique des Cancer du sein Pr. X Pivot CHRU Besançon Inca provided blood & tumour samples from ~ 150 paGents Basis ER+, HER-­‐ Inca provided blood & tumour samples from ~ 50 paGents Fr HER+ Inca leads the project for this subgroup ~ 150 paGents 75 Whole Genomes 30 Whole Exomes 75 RNASeq Uk Triple Neg. Total sequenced & analyzed 560 tumours (~140 from INCa) Études sein INCa Mai 2006 Juillet 2010 PHARE 3400 patientes HER2+ Pharmacogénétique PHARE 650 patientes HER2 + sang • Suivi clinique pour 5 ans • Echantillon de sang (optionnel) Mai 2009 Octobre 2011 SIGNAL 3000 patientes HER2+ sang 6000 patientes HER2- sang SIGNAL2-ICGC 500 tumeurs HER2+ 2000 tumeurs HER2- Mai 2009 ? 2014 • Suivi clinique pour 5 ans • Questionnaire épidémiologique SIGNAL • Echantillon de sang + • Tumeur pour SIGNAL2-ICGC-BASIS doi:10.1038/nature17676 Landscape of somatic mutations in 560 breast cancer whole-genome sequences Serena Nik-Zainal1,2, Helen Davies1, Johan Staaf3, Manasa Ramakrishna1, Dominik Glodzik1, Xueqing Zou1, Inigo Martincorena1, Ludmil B. Alexandrov1,4,5, Sancha Martin1, David C. Wedge1, Peter Van Loo1,6, Young Seok Ju1, Marcel Smid7, Arie B. Brinkman8, Sandro Morganella9, Miriam R. Aure10,11, Ole Christian Lingjærde11,12, Anita Langerød10,11, Markus Ringnér3, Sung-Min Ahn13, Sandrine Boyault14, Jane E. Brock15, Annegien Broeks16, Adam Butler1, Christine Desmedt17, Luc Dirix18, Serge Dronov1, Aquila Fatima19, John A. Foekens7, Moritz Gerstung1, Gerrit K. J. Hooijer20, Se Jin Jang21, David R. Jones1, Hyung-Yong Kim22, Tari A. King23, Savitri Krishnamurthy24, Hee Jin Lee21, Jeong-Yeon Lee25, Yilong Li1, Stuart McLaren1, Andrew Menzies1, Ville Mustonen1, Sarah O’Meara1, Iris Pauporté26, Xavier Pivot27, Colin A. Purdie28, Keiran Raine1, Kamna Ramakrishnan1, F. Germán Rodríguez-González7, Gilles Romieu29, Anieta M. Sieuwerts7, Peter T. Simpson30, Rebecca Shepherd1, Lucy Stebbings1, Olafur A. Stefansson31, Jon Teague1, Stefania Tommasi32, Isabelle Treilleux33, Gert G. Van den Eynden18,34, Peter Vermeulen18,34, Anne Vincent-Salomon35, Lucy Yates1, Carlos Caldas36, Laura van’t Veer16, Andrew Tutt37,38, Stian Knappskog39,40, Benita Kiat Tee Tan41,42, Jos Jonkers16, Åke Borg3, Naoto T. Ueno24, Christos Sotiriou17, Alain Viari43,44, P. Andrew Futreal1,45, Peter J. Campbell1, Paul N. Span46, Steven Van Laere18, Sunil R. Lakhani30,47, Jorunn E. Eyfjord31, Alastair M. Thompson28,48, Ewan Birney9, Hendrik G. Stunnenberg8, Marc J. van de Vijver20, John W. M. Martens7, Anne-Lise Børresen-Dale10,11, Andrea L. Richardson15,19, Gu Kong22, Gilles Thomas44 & Michael R. Stratton1 ARTICLE RESEARCH We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement B 560 whole-genome signatures. ThreeArearrangement signatures, characterized by tandem duplications or deletions, appear associated with sequenced breast cancers defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across 34% exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer. ER pos 320 The mutational theory of cancer proposes that changes in DNA sequence, termed ‘driver’ mutations, confer proliferative advantage on a cell, leading to outgrowth of a neoplastic clone1. Some driver mutations are inherited in the germline, but most arise in 36% 10% 21% somatic cells during the lifetime of the cancer patient, together with ERmutations neg not implicated in cancer development1. many ‘passenger’ Multiple mutational 167processes, including endogenous and exogenous mutagen exposures, aberrant DNA editing, replication errors substitutions 46 27 Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. 2East Anglian Medical Genetics Service, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 9NB, UK. rearrangements Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund SE-223 81, Sweden. 4Theoretical Biology and Biophysics (T-6), Los Alamos National Laboratory, of Los Alamos, NM 87545, New Mexico, USA. 5Center for Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. 6Department of Human Genetics, University indels Leuven, B-3000 Leuven, Belgium. 7Department of Medical Oncology, Erasmus MC Cancer Institute and Cancer Genomics Netherlands, Erasmus University Medical Center, Rotterdam 3015CN, The Netherlands. 8Radboud University, Department of Molecular Biology, Faculties of Science and Medicine, 6525GA Nijmegen, The Netherlands. 9European Molecular Biology Laboratory, copy number aberrations European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK. 10Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo 0310, Norway. 11K. G. Jebsen Centre for Breast Cancer Research, Institute for Clinical Medicine, University of Oslo, Oslo 0310, Norway. 12Department of Computer Science, University of Oslo, Oslo, Norway. 13Gachon Institute of Genome Medicine and Science, Gachon University Gil Medical Center, Incheon, South Korea. 14Translational Research Chr12 Lab, Centre Léon Bérard, 28, rue Laënnec, 69373 Lyon Cedex Chr1 08, France. 15Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA. 16The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. 17Breast Cancer Translational Research Laboratory, Université Libre de Bruxelles, Institut Jules Bordet, Bd de Waterloo 121, B-1000 Antwerp, Antwerp, 2.0e+07 Belgium. 19Dana-Farber Cancer Brussels, Belgium. 18Translational Cancer Research Unit, Center for Oncological Research, Faculty of Medicine and Health Sciences, University of 0.0e+00 4.0e+07 6.0e+07 8.0e+07 0.0e+00 5.0e+07 1.0e+08 1.5e+08 2.0e+08 2.5e+08 Chr13 Institute, Boston, Massachusetts 02215, USA. 20Department of Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 21Department of Pathology, Asan Chr2 Medical Center, College of Medicine, Ulsan University, Ulsan, South Korea. 22Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, South Korea. 23Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA. 24Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson 0e+00 2e+07 4e+07 6e+07 26 and Biopharmaceutical Research Cancer Center, 1515 Holcombe Texas 77030, USA. 25Institute 0.0e+00 5.0e+07 Boulevard., Houston, 1.0e+08 1.5e+08for Bioengineering2.0e+08 2.5e+08 (IBBR), Hanyang University, Seoul, South Korea. Institut 0 500 HER2 pos 1.0e+08 1.2e+08 500 3 0 1 27 0 500 0 500 0 ARTICLE Chr14 8e+07 1e+08 ARTICLE ER positive doi:10.1038/nature17676 Landscape of somatic mutations in 560 breast cancer whole-genome sequences b Fraction of samples with driver 100 80 60 40 20 0 Serena Nik-Zainal1,2, Helen Davies1, Johan Staaf3, Manasa Ramakrishna1, Dominik Glodzik1, Xueqing Zou1, Inigo Martincorena1, Ludmil B. Alexandrov1,4,5, Sancha Martin1, David C. Wedge1, Peter Van Loo1,6, Young Seok Ju1, Marcel Smid7, Arie B. Brinkman8, Sandro Morganella9, Miriam R. Aure10,11, Ole Christian Lingjærde11,12, Anita Langerød10,11, Markus Ringnér3, Sung-Min Ahn13, Sandrine Boyault14, Jane E. Brock15, Annegien Broeks16, Adam Butler1, Christine Desmedt17, Luc Dirix18, Serge Dronov1, Aquila Fatima19, John A. Foekens7, Moritz Gerstung1, Gerrit K. J. Hooijer20, Se Jin Jang21, David R. Jones1, Hyung-Yong Kim22, Tari A. King23, Savitri Krishnamurthy24, Hee Jin Lee21, Jeong-Yeon Lee25, Yilong Li1, Stuart McLaren1, Andrew Menzies1, Ville Mustonen1, Sarah O’Meara1, Iris Pauporté26, Xavier Pivot27, Colin A. Purdie28, Keiran Raine1, Kamna Ramakrishnan1, F. Germán Rodríguez-González7, Gilles Romieu29, Anieta M. Sieuwerts7, Peter T. Simpson30, Rebecca Shepherd1, Lucy Stebbings1, Olafur A. Stefansson31, Jon Teague1, Stefania Tommasi32, Isabelle Treilleux33, Gert G. Van den Eynden18,34, Peter Vermeulen18,34, Anne Vincent-Salomon35, Lucy Yates1, Carlos Caldas36, Laura van’t Veer16, Andrew Tutt37,38, Stian Knappskog39,40, Benita Kiat Tee Tan41,42, Jos Jonkers16, Åke Borg3, Naoto T. Ueno24, Christos Sotiriou17, Alain Viari43,44, P. Andrew Futreal1,45, Peter J. Campbell1, Paul N. Span46, Steven Van Laere18, Sunil R. Lakhani30,47, Jorunn E. Eyfjord31, Alastair M. Thompson28,48, Ewan Birney9, Hendrik G. Stunnenberg8, Marc J. van de Vijver20, John W. M. Martens7, Anne-Lise Børresen-Dale10,11, Andrea L. Richardson15,19, Gu Kong22, Gilles Thomas44 & Michael R. Stratton1 ER positive We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer. The mutational theory of cancer proposes that changes in DNA sequence, termed ‘driver’ mutations, confer proliferative advantage on a cell, leading to outgrowth of a neoplastic clone1. Some driver mutations are inherited in the germline, but most arise in 1 somatic cells during the lifetime of the cancer patient, together with many ‘passenger’ mutations not implicated in cancer development1. Multiple mutational processes, including endogenous and exogenous mutagen exposures, aberrant DNA editing, replication errors RESEARCH ARTICLE Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. 2East Anglian Medical Genetics Service, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 9NB, UK. Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund SE-223 81, Sweden. 4Theoretical Biology and Biophysics (T-6), Los Alamos National Laboratory, Los Alamos, NM 87545, New Mexico, USA. 5Center for Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. 6Department of Human Genetics, University of Leuven, B-3000 Leuven, Belgium. 7Department of Medical Oncology, Erasmus MC Cancer Institute and Cancer Genomics Netherlands, Erasmus University Medical Center, Rotterdam 3015CN, The Netherlands. 8Radboud University, Department of Molecular Biology, Faculties of Science and Medicine, 6525GA Nijmegen, The Netherlands. 9European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK. 10Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo 0310, Norway. 11K. G. Jebsen Centre for Breast Cancer Research, Institute for Clinical Medicine, University of Oslo, Oslo 0310, Norway. 12Department of Computer Science, University of Oslo, Oslo, Norway. 13Gachon Institute of Genome Medicine and Science, Gachon University Gil Medical Center, Incheon, South Korea. 14Translational Research Lab, Centre Léon Bérard, 28, rue Laënnec, 69373 Lyon Cedex 08, France. 15Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA. 16The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. 17Breast Cancer Translational Research Laboratory, Université Libre de Bruxelles, Institut Jules Bordet, Bd de Waterloo 121, B-1000 Brussels, Belgium. 18Translational Cancer Research Unit, Center for Oncological Research, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium. 19Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA. 20Department of Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 21Department of Pathology, Asan Medical Center, College of Medicine, Ulsan University, Ulsan, South Korea. 22Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, South Korea. 23Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA. 24Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard., Houston, Texas 77030, USA. 25Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, South Korea. 26Institut National du Cancer, Research Division, Clinical Research Department, 52 avenue Morizet, 92513 Boulogne-Billancourt, France. 27University Hospital of Minjoz, INSERM UMR 1098, Bd Fleming, Besançon 25000, France. 28Pathology Department, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. 29Oncologie Sénologie, ICM Institut Régional du Cancer, Montpellier, France. 30 The University of Queensland, UQ Centre for Clinical Research and School of Medicine, Brisbane, Queensland 4059, Australia. 31Cancer Research Laboratory, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland. 32IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy. 33Department of Pathology, Centre Léon Bérard, 28 rue Laënnec, 69373 Lyon Cédex 08, France. 34 Department of Pathology, GZA Hospitals Sint-Augustinus, Antwerp, Belgium. 35Institut Curie, Department of Pathology and INSERM U934, 26 rue d’Ulm, 75248 Paris Cedex 05, France. 36Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. 37Breast Cancer Now Toby Robin’s Research Unit, King’s College London, London SE1 9RT, UK. 38Breast Cancer Now Toby Robin’s Research Centre, Institute of Cancer Research, London SW3 6JB, UK. 39Department of Clinical Science, University of Bergen, 5020 Bergen, Norway. 40Department of Oncology, Haukeland University Hospital, 5021 Bergen, Norway. 41National Cancer Centre Singapore, 11 Hospital Drive, 169610, Singapore. 42Singapore General Hospital, Outram Road, 169608, Singapore. 43Equipe Erable, INRIA Grenoble-Rhône-Alpes, 655, Avenue de l’Europe, 38330 Montbonnot-Saint Martin, France. 44Synergie Lyon Cancer, Centre Léon Bérard, 28 rue Laënnec, Lyon Cedex 08, France. 45Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, Texas 77230, USA. 46Department of Radiation Oncology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen 6525GA, The Netherlands. 47Pathology Queensland, The Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia. 48Department of Surgical Oncology, University of Texas MD Anderson Cancer Center, 1400 Pressler Street, Houston, Texas 77030, USA. 3 a 80,000 5,000 1,200 20 Substitutions * Indels Rearrangements 0 0 M O N T H 2 0 1 6 | VO L 0 0 0 | NAT U R E | 1 Driver mutations ER positive b * Fraction of samples with driver 100 80 60 40 20 0 Enrichment log P value –10 0 10 ER negative Fraction of samples with driver 0 20 40 60 80 100 Enrichment log P value –10 0 10 TP53 PIK3CA MYC CCND1 PTEN ERBB2 ZNF703/FGFR1 GATA3 RB1 MAP3K1 MAP2K4 ZNF217 CDH1 MLL3 ARID1B CDKN2A MLLT4 AKT1 FBXW7 ARID1A CCND3 CBFB MDM2 CCNE1 CDKN2B NCOR1 SF3B1 SPEN TBX3 IGF1R BRCA2 NF1 PIK3R1 EGFR KRAS ESR1 FOXA1 MED23 CDK6 NOTCH2 AKT2 BRCA1 CTCF KDM6A SETD2 CREBBP DNMT3A FOXP1 MLL2 RUNX1 USP9X XBP1 PDGFRA ATR ERBB3 FGFR2 PALB2 RHOA SMAD4 ATM ATRX AXIN1 BCOR 10 CDKN1B CUX1 GNAS MLH1 NOTCH1 PHF6 SMARCA4 STAG2 ZFP36L1 APC CASP8 CBLB CNOT3 ECT2L MEN1 MSH2 NRAS PBRM1 PMS2 STK11 TET2 ASXL1 BRAF BUB1B CIC ERCC4 HRAS NF2 PRDM1 PREX2 Figure 1 | Cohort and catalogue of somatic mutations in 560 breast cancers. a, Catalogue of base substitutions, insertions/deletions, rearrangements and driver mutations in 560 breast cancers (sorted by total substitution burden). Indel axis limited to 5,000(*). b, Complete list of curated driver genes sorted by frequency (descending). Fraction of ERpositive (left, total 366) and ER-negative (right, total 194) samples carrying a mutation in the relevant driver gene presented in grey. log P value of enrichment of each driver gene towards the ER-positive or ER-negative cohort is provided in black. Highlighted in green are genes for which there is new or further evidence supporting these as novel breast cancer genes. and defective DNA maintenance, are responsible for generating these mutations1–3. Over the past five decades, several waves of technology have advanced the characterization of mutations in cancer genomes. Karyotype analysis revealed rearranged chromosomes and copy number alterations. Subsequently, loss of heterozygosity analysis, hybridization of cancer-derived DNA to microarrays and other approaches provided higher resolution insights into copy number changes4–8. Recently, DNA sequencing has enabled systematic characterization of the full repertoire of mutation types including base substitutions, small insertions/ ER negative Fraction of samples with driver 0 20 40 60 80 100 ER negative the charac analysis r alterations of cancer-d higher reso sequencin toire of mu deletions, substantia processes o As for m genome se there has b intronic an to the mol of activatin genes/prot as observe role of dri genome20, human dis the possib was highlig TERT gene the somat question h on genom tion mutat signatures Here we address th hensive un mutations Cancer g The whol tissue fro sequence We detecte and 77,695 of each bet Transcripto ber and DN To ident and indels constitutin clustering genes were (MED23, F tions indic a dominan ARTICLE ER positive b doi:10.1038/nature17676 Landscape of somatic mutations in 560 breast cancer whole-genome sequences Fraction of samples with driver 100 80 60 40 20 0 Serena Nik-Zainal1,2, Helen Davies1, Johan Staaf3, Manasa Ramakrishna1, Dominik Glodzik1, Xueqing Zou1, Inigo Martincorena1, Ludmil B. Alexandrov1,4,5, Sancha Martin1, David C. Wedge1, Peter Van Loo1,6, Young Seok Ju1, Marcel Smid7, Arie B. Brinkman8, Sandro Morganella9, Miriam R. Aure10,11, Ole Christian Lingjærde11,12, Anita Langerød10,11, Markus Ringnér3, Sung-Min Ahn13, Sandrine Boyault14, Jane E. Brock15, Annegien Broeks16, Adam Butler1, Christine Desmedt17, Luc Dirix18, Serge Dronov1, Aquila Fatima19, John A. Foekens7, Moritz Gerstung1, Gerrit K. J. Hooijer20, Se Jin Jang21, David R. Jones1, Hyung-Yong Kim22, Tari A. King23, Savitri Krishnamurthy24, Hee Jin Lee21, Jeong-Yeon Lee25, Yilong Li1, Stuart McLaren1, Andrew Menzies1, Ville Mustonen1, Sarah O’Meara1, Iris Pauporté26, Xavier Pivot27, Colin A. Purdie28, Keiran Raine1, Kamna Ramakrishnan1, F. Germán Rodríguez-González7, Gilles Romieu29, Anieta M. Sieuwerts7, Peter T. Simpson30, Rebecca Shepherd1, Lucy Stebbings1, Olafur A. Stefansson31, Jon Teague1, Stefania Tommasi32, Isabelle Treilleux33, Gert G. Van den Eynden18,34, Peter Vermeulen18,34, Anne Vincent-Salomon35, Lucy Yates1, Carlos Caldas36, Laura van’t Veer16, Andrew Tutt37,38, Stian Knappskog39,40, Benita Kiat Tee Tan41,42, Jos Jonkers16, Åke Borg3, Naoto T. Ueno24, Christos Sotiriou17, Alain Viari43,44, P. Andrew Futreal1,45, Peter J. Campbell1, Paul N. Span46, Steven Van Laere18, Sunil R. Lakhani30,47, Jorunn E. Eyfjord31, Alastair M. Thompson28,48, Ewan Birney9, Hendrik G. Stunnenberg8, Marc J. van de Vijver20, John W. M. Martens7, Anne-Lise Børresen-Dale10,11, Andrea L. Richardson15,19, Gu Kong22, Gilles Thomas44 & Michael R. Stratton1 ER positive We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer. The mutational theory of cancer proposes that changes in DNA sequence, termed ‘driver’ mutations, confer proliferative advantage on a cell, leading to outgrowth of a neoplastic clone1. Some driver mutations are inherited in the germline, but most arise in A 30 PIK3CA 25 GATA3 promoter PLEKHS1 TP53 20 15 10 intergenic intron GPR126 PIK3CA SF3B1 GATA3 intergenic TP53 AKT1 XBP1 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 X Y 0 0 M O N T H 2 0 1 6 | VO L 0 0 0 | NAT U R E | 1 chromosome B lncRNA MALAT1 lncRNA NEAT1 sub indel ● ●● ●● 65,190,000 ● ● ● ● ● ● ●●●●●● ● ●● ● ● ● ● ●●●● ● ● ●● ●● ●● ● 65,200,000 chr11 coordinate (Mbp) ● ●● ● ● ● 65,210,000 sub –10 0 10 TP53 PIK3CA MYC CCND1 PTEN ERBB2 ZNF703/FGFR1 GATA3 RB1 MAP3K1 MAP2K4 ZNF217 CDH1 MLL3 ARID1B CDKN2A MLLT4 AKT1 FBXW7 ARID1A CCND3 CBFB MDM2 CCNE1 CDKN2B NCOR1 SF3B1 SPEN TBX3 IGF1R BRCA2 NF1 PIK3R1 EGFR KRAS ESR1 FOXA1 MED23 CDK6 NOTCH2 AKT2 BRCA1 CTCF KDM6A SETD2 CREBBP DNMT3A FOXP1 MLL2 RUNX1 USP9X XBP1 PDGFRA ATR ERBB3 FGFR2 PALB2 RHOA SMAD4 ATM ATRX AXIN1 BCOR CDKN1B CUX1 GNAS MLH1 NOTCH1 PHF6 SMARCA4 STAG2 ZFP36L1 APC CASP8 CBLB CNOT3 ECT2L MEN1 MSH2 NRAS PBRM1 PMS2 STK11 TET2 ASXL1 BRAF BUB1B CIC ERCC4 HRAS NF2 PRDM1 PREX2 ARTICLE RESEARCH somatic cells during the lifetime of the cancer patient, together with many ‘passenger’ mutations not implicated in cancer development1. Multiple mutational processes, including endogenous and exogenous mutagen exposures, aberrant DNA editing, replication errors Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. 2East Anglian Medical Genetics Service, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 9NB, UK. Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund SE-223 81, Sweden. 4Theoretical Biology and Biophysics (T-6), Los Alamos National Laboratory, Los Alamos, NM 87545, New Mexico, USA. 5Center for Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. 6Department of Human Genetics, University of Leuven, B-3000 Leuven, Belgium. 7Department of Medical Oncology, Erasmus MC Cancer Institute and Cancer Genomics Netherlands, Erasmus University Medical Center, Rotterdam 3015CN, The Netherlands. 8Radboud University, Department of Molecular Biology, Faculties of Science and Medicine, 6525GA Nijmegen, The Netherlands. 9European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK. 10Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo 0310, Norway. 11K. G. Jebsen Centre for Breast Cancer Research, Institute for Clinical Medicine, University of Oslo, Oslo 0310, Norway. 12Department of Computer Science, University of Oslo, Oslo, Norway. 13Gachon Institute of Genome Medicine and Science, Gachon University Gil Medical Center, Incheon, South Korea. 14Translational Research Lab, Centre Léon Bérard, 28, rue Laënnec, 69373 Lyon Cedex 08, France. 15Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA. 16The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. 17Breast Cancer Translational Research Laboratory, Université Libre de Bruxelles, Institut Jules Bordet, Bd de Waterloo 121, B-1000 Brussels, Belgium. 18Translational Cancer Research Unit, Center for Oncological Research, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium. 19Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA. 20Department of Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 21Department of Pathology, Asan Medical Center, College of Medicine, Ulsan University, Ulsan, South Korea. 22Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, South Korea. 23Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA. 24Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard., Houston, Texas 77030, USA. 25Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, South Korea. 26Institut National du Cancer, Research Division, Clinical Research Department, 52 avenue Morizet, 92513 Boulogne-Billancourt, France. 27University Hospital of Minjoz, INSERM UMR 1098, Bd Fleming, Besançon 25000, France. 28Pathology Department, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. 29Oncologie Sénologie, ICM Institut Régional du Cancer, Montpellier, France. 30 The University of Queensland, UQ Centre for Clinical Research and School of Medicine, Brisbane, Queensland 4059, Australia. 31Cancer Research Laboratory, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland. 32IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy. 33Department of Pathology, Centre Léon Bérard, 28 rue Laënnec, 69373 Lyon Cédex 08, France. 34 Department of Pathology, GZA Hospitals Sint-Augustinus, Antwerp, Belgium. 35Institut Curie, Department of Pathology and INSERM U934, 26 rue d’Ulm, 75248 Paris Cedex 05, France. 36Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. 37Breast Cancer Now Toby Robin’s Research Unit, King’s College London, London SE1 9RT, UK. 38Breast Cancer Now Toby Robin’s Research Centre, Institute of Cancer Research, London SW3 6JB, UK. 39Department of Clinical Science, University of Bergen, 5020 Bergen, Norway. 40Department of Oncology, Haukeland University Hospital, 5021 Bergen, Norway. 41National Cancer Centre Singapore, 11 Hospital Drive, 169610, Singapore. 42Singapore General Hospital, Outram Road, 169608, Singapore. 43Equipe Erable, INRIA Grenoble-Rhône-Alpes, 655, Avenue de l’Europe, 38330 Montbonnot-Saint Martin, France. 44Synergie Lyon Cancer, Centre Léon Bérard, 28 rue Laënnec, Lyon Cedex 08, France. 45Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, Texas 77230, USA. 46Department of Radiation Oncology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen 6525GA, The Netherlands. 47Pathology Queensland, The Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia. 48Department of Surgical Oncology, University of Texas MD Anderson Cancer Center, 1400 Pressler Street, Houston, Texas 77030, USA. 3 −log10(pval) 1 Enrichment log P value ● ● ●● ●● ● ● ● indel ● ●● ● ● ● ●●● ●● 65,266,000 ● ● ●● ●● ● ● ● ● ● ● ●● ● ● 65,270,000 chr11 coordinate (Mbp) ● ● ● ● ●●● ● 65,274,000 ER negative Fraction of samples with driver 0 20 40 60 80 100 ER negative the charac analysis r alterations of cancer-d higher reso sequencin toire of mu deletions, substantia processes o As for m genome se there has b intronic an to the mol of activatin genes/prot as observe role of dri genome20, human dis the possib was highlig TERT gene the somat question h on genom tion mutat signatures Here we address th hensive un mutations Cancer g The whol tissue fro sequence We detecte and 77,695 of each bet Transcripto ber and DN To ident and indels constitutin clustering genes were (MED23, F tions indic a dominan 0.2 ARTICLE 0.1 0.2 Signature 8 0.1 Signature 26 doi:10.1038/nature17676 0.2 Landscape of somatic mutations in 560 breast cancer whole-genome sequences 0.1 Serena Nik-Zainal1,2, Helen Davies1, Johan Staaf3, Manasa Ramakrishna1, Dominik Glodzik1, Xueqing Zou1, Inigo Martincorena1, Ludmil B. Alexandrov1,4,5, Sancha Martin1, David C. Wedge1, Peter Van Loo1,6, Young Seok Ju1, Marcel Smid7, Arie B. Brinkman8, Sandro Morganella9, Miriam R. Aure10,11, Ole Christian Lingjærde11,12, Anita Langerød10,11, Markus Ringnér3, Sung-Min Ahn13, Sandrine Boyault14, Jane E. Brock15, Annegien Broeks16, Adam Butler1, Christine Desmedt17, Luc Dirix18, Serge Dronov1, Aquila Fatima19, John A. Foekens7, Moritz Gerstung1, Gerrit K. J. Hooijer20, Se Jin Jang21, David R. Jones1, Hyung-Yong Kim22, Tari A. King23, Savitri Krishnamurthy24, Hee Jin Lee21, Jeong-Yeon Lee25, Yilong Li1, Stuart McLaren1, Andrew Menzies1, Ville Mustonen1, Sarah O’Meara1, Iris Pauporté26, Xavier Pivot27, Colin A. Purdie28, Keiran Raine1, Kamna Ramakrishnan1, F. Germán Rodríguez-González7, Gilles Romieu29, Anieta M. Sieuwerts7, Peter T. Simpson30, Rebecca Shepherd1, Lucy Stebbings1, Olafur A. Stefansson31, Jon Teague1, Stefania Tommasi32, Isabelle Treilleux33, Gert G. Van den Eynden18,34, Peter Vermeulen18,34, Anne Vincent-Salomon35, Lucy Yates1, Carlos Caldas36, Laura van’t Veer16, Andrew Tutt37,38, Stian Knappskog39,40, Benita Kiat Tee Tan41,42, Jos Jonkers16, Åke Borg3, Naoto T. Ueno24, Christos Sotiriou17, Alain Viari43,44, P. Andrew Futreal1,45, Peter J. Campbell1, Paul N. Span46, Steven Van Laere18, Sunil R. Lakhani30,47, Jorunn E. Eyfjord31, Alastair M. Thompson28,48, Ewan Birney9, Hendrik G. Stunnenberg8, Marc J. van de Vijver20, John W. M. Martens7, Anne-Lise Børresen-Dale10,11, Andrea L. Richardson15,19, Gu Kong22, Gilles Thomas44 & Michael R. Stratton1 0.2 Signature 30 0.1 Signature 5 100,000 b 0 100% RESEARCH ARTICLE We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended genome rearrangements revealed T>G twelve base substitution and six rearrangement C>A toC>G C>T T>A andT>C C>A C>G C>T signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with 0.2 0.2 defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient Signature 18 across Signature BRCA1 or BRCA2 function, the cause 1 of the third is unknown. This analysis of all classes of somatic mutation exons, introns 0.1 and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and 0.1 progresses towards a comprehensive account of the somatic genetic basis of breast cancer. a The mutational theory of cancer proposes that changes in DNA sequence, termed0.2 ‘driver’ mutations, confer proliferative advantage on a cell, leading to outgrowth of a neoplastic clone1. Some Signature 2 driver mutations are inherited in the germline, but most arise in 0.1 T>A T>C T>G somatic cells during the lifetime of the cancer patient, together with many ‘passenger’ mutations not implicated in cancer development1. 0.2 Multiple mutational processes, including endogenous Signature 17and exogenous mutagen exposures, aberrant DNA editing, replication errors 0.1 1 Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. 2East Anglian Medical Genetics Service, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 9NB, UK. Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund SE-223 81, Sweden. 4Theoretical Biology and Biophysics (T-6), Los Alamos National Laboratory, Los Alamos, NM 87545, New Mexico, USA. 5Center for Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. 6Department of Human Genetics, University of Leuven, B-3000 Leuven, Belgium. 7Department of Medical Oncology, Erasmus MC Cancer Institute and Cancer Genomics Netherlands, Erasmus University Medical Center, Rotterdam 3015CN, The Netherlands. 8Radboud University, Department of Molecular Biology, Faculties of Science and Medicine, 6525GA Nijmegen, The Netherlands. 9European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK. 10Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo 0310, Norway. 11K. G. Jebsen Centre for Breast Cancer Research, Institute for Clinical Medicine, University of Oslo, Oslo 0310, Norway. 12Department of Computer Science, University of Oslo, Oslo, Norway. 13Gachon Institute of Genome Medicine and Science, Gachon University Gil Medical Center, Incheon, South Korea. 14Translational Research Lab, Centre Léon Bérard, 28, rue Laënnec, 69373 Lyon Cedex 08, France. 15Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA. 16The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. 17Breast Cancer Translational Research Laboratory, Université Libre de Bruxelles, Institut Jules Bordet, Bd de Waterloo 121, B-1000 Brussels, Belgium. 18Translational Cancer Research Unit, Center for Oncological Research, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium. 19Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA. 20Department of Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 21Department of Pathology, Asan Medical Center, College of Medicine, Ulsan University, Ulsan, South Korea. 22Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, South Korea. 23Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA. 24Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard., Houston, Texas 77030, USA. 25Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, South Korea. 26Institut National du Cancer, Research Division, Clinical Research Department, 52 avenue Morizet, 92513 Boulogne-Billancourt, France. 27University Hospital of Minjoz, INSERM UMR 1098, Bd Fleming, Besançon 25000, France. 28Pathology Department, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. 29Oncologie Sénologie, ICM Institut Régional du Cancer, Montpellier, France. 30 The University of Queensland, UQ Centre for Clinical Research and School of Medicine, Brisbane, Queensland 4059, Australia. 31Cancer Research Laboratory, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland. 32IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy. 33Department of Pathology, Centre Léon Bérard, 28 rue Laënnec, 69373 Lyon Cédex 08, France. 34 Department of Pathology, GZA Hospitals Sint-Augustinus, Antwerp, Belgium. 35Institut Curie, Department of Pathology and INSERM U934, 26 rue d’Ulm, 75248 Paris Cedex 05, France. 36Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. 37Breast Cancer Now Toby Robin’s Research Unit, King’s College London, London SE1 9RT, UK. 38Breast Cancer Now Toby Robin’s Research Centre, Institute of Cancer Research, London SW3 6JB, UK. 39Department of Clinical Science, University of Bergen, 5020 Bergen, Norway. 40Department of Oncology, Haukeland University Hospital, 5021 Bergen, Norway. 41National Cancer Centre Singapore, 11 Hospital Drive, 169610, Singapore. 42Singapore General Hospital, Outram Road, 169608, Singapore. 43Equipe Erable, INRIA Grenoble-Rhône-Alpes, 655, Avenue de l’Europe, 38330 Montbonnot-Saint Martin, France. 44Synergie Lyon Cancer, Centre Léon Bérard, 28 rue Laënnec, Lyon Cedex 08, France. 45Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, Texas 77230, USA. 46Department of Radiation Oncology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen 6525GA, The Netherlands. 47Pathology Queensland, The Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia. 48Department of Surgical Oncology, University of Texas MD Anderson Cancer Center, 1400 Pressler Street, Houston, Texas 77030, USA. 3 0.2 Signature 0.1 13 0.2 0.1 0.2 0.1 Signature 3 Signature 8 0.2 0.1 0.2 0.1 0.2 0.1 Signature 6 Signature 20 Signature 26 Previously identified Mutational signatures in breast cancers 0 0 M O N T H 2 0 1 6 | VO L 0 0 0 | NAT U R E | 1 0.2 0.1 b Signature 5 The WDR74 promoter showed base substitutions and 0% ind (q value 4.6 × 10−3) forming a cluster of overlapping mutation Signatures 1 5 sequence 2 13 6 driver 20 26 mutations 3 8 18 in 17 WDR74 30 (Fig. 2a). Coding have not b reported. No differences were observed in WDR74 transcript le c 100 80 with WDR74 promoter mutations compared to th between cancers Percentage 60 without. Nevertheless, the pattern of this non-coding mutation clus of samples 40 with overlapping and different mutation types, is more compatible w 20 the possibility of the mutations being drivers. 100,000 Two long10,000 non-coding RNAs, MALAT1 (q value 8.7 × 10−11, as pr Mutation 12 −2 ouslycount reported 1,000 ) and NEAT1 (q value 2.1 × 10 ) were enriched w per sample Transcript mutations. levels were not significantly different betw 100 10 mutated and non-mutated samples. Whether these mutations are d 1 ers or result from local hypermutability is unclear. 1 2 13 3 8 5 18 17 6 20 26 30 0.2 Signature 30 0.1 100,000 Previously identified in Novel other cancer types signatures Mutational processes generating somatic parti Figure 3 | Extraction and contributions of base mutations substitutionimprint signatures 2,2 lar patterns of mutations on cancer genomes, termed signatures in 560 breast cancers. a, Twelve mutation signatures extracted using non25 Applying a mathematical approach extract negative matrix factorization. Each signature isto ordered by mutational mutation classsig (C>A/G>T, C>G/G>C, C>T/G>A, T>A/A>T, T>C/A>G, T>G/A tures previously revealed five base-substitution signatures in br >C), takingsignatures immediate1, flanking into account. For each cancer: 2, 3, 8 sequence and 13 (refs 2, 24). Using thisclass, method mutations are ordered by 5′ base (A, C, G, T) first before 3′ base (A, C, the 560 cases revealed twelve signatures, including those previou G, T). b, The spectrum of base substitution signatures within 560 breast observed and a further seven, of which five have formerly been detec cancers. Mutation signatures are ordered (and coloured) according to ARTICLE doi:10.1038/nature17676 Landscape of somatic mutations in 560 breast cancer whole-genome sequences Serena Nik-Zainal1,2, Helen Davies1, Johan Staaf3, Manasa Ramakrishna1, Dominik Glodzik1, Xueqing Zou1, Inigo Martincorena1, Ludmil B. Alexandrov1,4,5, Sancha Martin1, David C. Wedge1, Peter Van Loo1,6, Young Seok Ju1, Marcel Smid7, Arie B. Brinkman8, Sandro Morganella9, Miriam R. Aure10,11, Ole Christian Lingjærde11,12, Anita Langerød10,11, Markus Ringnér3, Sung-Min Ahn13, Sandrine Boyault14, Jane E. Brock15, Annegien Broeks16, Adam Butler1, Christine Desmedt17, Luc Dirix18, Serge Dronov1, Aquila Fatima19, John A. Foekens7, Moritz Gerstung1, Gerrit K. J. Hooijer20, Se Jin Jang21, David R. Jones1, Hyung-Yong Kim22, Tari A. King23, Savitri Krishnamurthy24, Hee Jin Lee21, Jeong-Yeon Lee25, Yilong Li1, Stuart McLaren1, Andrew Menzies1, Ville Mustonen1, Sarah O’Meara1, Iris Pauporté26, Xavier Pivot27, Colin A. Purdie28, Keiran Raine1, Kamna Ramakrishnan1, F. Germán Rodríguez-González7, Gilles Romieu29, Anieta M. Sieuwerts7, Peter T. Simpson30, Rebecca Shepherd1, Lucy Stebbings1, Olafur A. Stefansson31, Jon Teague1, Stefania Tommasi32, Isabelle Treilleux33, Gert G. Van den Eynden18,34, Peter Vermeulen18,34, Anne Vincent-Salomon35, Lucy Yates1, Carlos Caldas36, Laura van’t Veer16, Andrew Tutt37,38, Stian Knappskog39,40, Benita Kiat Tee Tan41,42, Jos Jonkers16, Åke Borg3, Naoto T. Ueno24, Christos Sotiriou17, Alain Viari43,44, P. Andrew Futreal1,45, Peter J. Campbell1, Paul N. Span46, Steven Van Laere18, Sunil R. Lakhani30,47, Jorunn E. Eyfjord31, Alastair M. Thompson28,48, Ewan Birney9, Hendrik G. Stunnenberg8, Marc J. van de Vijver20, John W. M. Martens7, Anne-Lise Børresen-Dale10,11, Andrea L. Richardson15,19, Gu Kong22, Gilles Thomas44 & Michael R. Stratton1 ARTICLE RESEARCH 6 20 26 3 8 18 17 30 inv trans trans Rearrangement signature 4 0% 60% 0% 60% 0% Rearrangement signature 5 60% 0% 60% 60% >10 Mb 1–10 Mb 0% 10–100 kb 0% 60% >10 Mb Rearrangement signature 6 1–10 kb 0% 1–10 Mb 0.20 100 kb–1 Mb 13 inv 1–10 kb 1–10 Mb 10–100 kb >10 Mb 100 kb–1 Mb 1–10 kb 1–10 Mb 10–100 kb >10 Mb 100 kb–1 Mb 1–10 kb 1–10 Mb 10–100 kb >10 Mb 100 kb–1 Mb 2 0% Rearrangement signature 3 100 kb–1 Mb 5 18 17 30 tds 1–10 kb 100 kb–1 Mb 10–100 kb 1–10 Mb 100 kb–1 Mb >10 Mb 1–10 Mb 1–10 kb >10 Mb 10–100 kb Signatures 1 8 tds 0% 1–10 Mb 1–10 kb >10 Mb 0.20 0.05 0.10 0.15 log replication Signatures 1 strand 5 2asymmetry 13 6 20 26 3 0.00 del 10–100 kb 0.00 0.05 0.10 0.15 log replication strand asymmetry trans 1–10 kb 1–10 Mb 10–100 kb >10 Mb 100 kb–1 Mb 1–10 kb 1–10 Mb 10–100 kb >10 Mb 100 kb–1 Mb 1–10 kb 1–10 Mb 10–100 kb >10 Mb 100 kb–1 Mb 0.00 0.00 0% APOBEC deaminase activity 60% APOBEC deaminase activity del 60% 10–100 kb 0.05 Associated with 0.05 mismatch repair deficiency mismatch repair deficiency Associated with trans Non-clustered rearrangements 60% 100 kb–1 Mb 0.10 inv 60% >10 Mb Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund SE-223 81, Sweden. Theoretical Biology and Biophysics (T-6), Los Alamos National Laboratory, Los Alamos, NM 87545, New Mexico, USA. 5Center for Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. 6Department of Human Genetics, University of Leuven, B-3000 Leuven, Belgium. 7Department of Medical Oncology, Erasmus MC Cancer Institute and Cancer Genomics Netherlands, Erasmus University Medical Center, Rotterdam 3015CN, The Netherlands. 8Radboud University, Department of Molecular Biology, Faculties of Science and Medicine, 6525GA Nijmegen, The Netherlands. 9European Molecular Biology Laboratory, Rearrangement European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK. 10Department of Cancer Genetics, Institute for Cancer Research, Oslo University Hospital, signature 3 of The Norwegian Radium Hospital, Oslo 0310, Norway. 11K. G. Jebsen Centre for Breast Cancer Research, Institute for Clinical Medicine, University of Oslo, Oslo 0310, Norway. 12Department Computer Science, University of Oslo, Oslo, Norway. 13Gachon Institute of Genome Medicine and Science, Gachon University Gil Medical Center, Incheon, South Korea. 14Translational Research Lab, Centre Léon Bérard, 28, rue Laënnec, 69373 Lyon Cedex 08, France. 15Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA. 16The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. 17Breast Cancer Translational Research Laboratory, Université Libre de Bruxelles, Institut Jules Bordet, Bd de Waterloo 121, B-1000 Rearrangement Cancer Brussels, Belgium. 18Translational Cancer Research Unit, Center for Oncological Research, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium. 19Dana-Farber 4 Asan Institute, Boston, Massachusetts 02215, USA. 20Department of Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 21Department of Pathology,signature Medical Center, College of Medicine, Ulsan University, Ulsan, South Korea. 22Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, South Korea. 23Memorial Sloan 24 Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA. Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard., Houston, Texas 77030, USA. 25Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, South Korea. 26Institut National du Cancer, Research Division, Clinical Research Department, 52 avenue Morizet, 92513 Boulogne-Billancourt, France. 27University Hospital of Minjoz, INSERM UMR 1098, Bd Fleming, Rearrangement Besançon 25000, France. 28Pathology Department, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. 29Oncologie Sénologie, ICM Institut Régional du Cancer, Montpellier, France. signature 5 30 The University of Queensland, UQ Centre for Clinical Research and School of Medicine, Brisbane, Queensland 4059, Australia. 31Cancer Research 10 Laboratory, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland. 32IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy. 33Department of Pathology, Centre Léon Bérard, 28 rue Laënnec, 69373 Lyon Cédex 08, France. 34 Department of Pathology, GZA Hospitals Sint-Augustinus, Antwerp, Belgium. 35Institut Curie, Department of Pathology and INSERM U934, 26 rue d’Ulm, 75248 Paris Cedex 05, France. 36Cancer London, Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. 37Breast Cancer Now Toby Robin’s Research Unit, King’s CollegeRearrangement 39 London SE1 9RT, UK. 38Breast Cancer Now Toby Robin’s Research Centre, Institute of Cancer Research, 10 London SW3 6JB, UK. Department of Clinical Science, University of Bergen, 5020 signature 6 Bergen, Norway. 40Department of Oncology, Haukeland University Hospital, 5021 Bergen, Norway. 41National Cancer Centre Singapore, 11 Hospital Drive, 169610, Singapore. 42Singapore General Hospital, Outram Road, 169608, Singapore. 43Equipe Erable, INRIA Grenoble-Rhône-Alpes, 655, Avenue de l’Europe, 38330 Montbonnot-Saint Martin, France. 44Synergie Lyon Cancer, Centre Léon Bérard, 28 rue Laënnec, Lyon Cedex 08, France. 45Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, Texas 77230, USA. 46Department of Radiation Oncology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen 6525GA, The Netherlands. 47Pathology Queensland, The Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia. 48Department of Surgical Oncology, University of Texas MD Anderson Cancer Center, 1400 Pressler Street, Houston, Texas 77030, USA. Rearrangement signature 2 1–10 kb Rearrangement 1 Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. 2East Anglian Medical Genetics Service, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2Associated 9NB, UK. signature 2 with Associated with 3 4 0% 1–10 Mb 0.10 tds 60% 100 kb–1 Mb Rearrangement somatic cells during the lifetime of the cancer patient, together with 1 signature many ‘passenger’ mutations not implicated in cancer development . 1 Multiple mutational processes, including endogenous and exogenous mutagen exposures, aberrant DNA editing, replication errors del 60%rearrangements Clustered Rearrangement signature del 1 inv 0% tds 1–10 kb The mutational theory of cancer proposes that changes in DNA sequence, termed ‘driver’ mutations, confer proliferative advan0.15 clone1. Some tage on a cell, leading to outgrowth of a neoplastic driver mutations are inherited in the germline, but most arise in 0.15 ARTICLE RESEARCH Non-clustered rearrangements Clustered rearrangements b 10–100 kb log10 transcription strand asymmetry log10 transcription strand asymmetry We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized 0.20 duplications or deletions, appear associated with aby tandem defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions the repertoire of cancer genes and mutational processes operating,band 0.20 a highlights progresses towards a comprehensive account of the somatic genetic basis of breast cancer. Rearrangement size 0 0 M O N T H 2 0 1 6 | VO L 0 0 0 | NAT U R E | 1 Figure 4 | Additional characteristics of base substitution signatures and signatures extracted using non-negative matrix factorization. Probability Rearrangement size novel rearrangement signatures in 560 breast cancers. a, Contrasting of rearrangement element on y axis. Rearrangement size on x axis. transcriptional strand and replication strand del, deletion; tds, tandem duplication; inv, inversion; trans, translocation. Figure 4 | Additional characteristics of asymmetry base substitution signatures and asymmetry signatures extracted using non-negative matrix factorization. Probability between twelve in base substitution signatures. b, Six rearrangement novel rearrangement signatures 560 breast cancers. a, Contrasting of rearrangement element on y axis. Rearrangement size on x axis. transcriptional strand asymmetry and replication strand asymmetry del, deletion; tds, tandem duplication; inv, inversion; trans, translocation. between twelve base substitution signatures. b, Six rearrangement there were nine locations at which recurrence of tandem duplications signatures 4 and 6 (Fig. 4b). Cancers with tandem duplications or deletions was found across the breast cancers and which often showed multiple, of rearrangement signatures 1, 3 and 5 did not usually demonstrate there were nine locations at which recurrenceinof tandem duplications and 6kataegis. (Fig. 4b).However, Cancers with tandem duplications deletions of kataegis as nested tandem duplications individual cases (Extendedsignatures Data Fig.48). there must be additionalordeterminants was found acrossThese the breast cancers and which often showed of duplication rearrangement signatures 1, 3 and 5 didare notassociated usually demonstrate may be mutational hotspots specific formultiple, these tandem only 2% of rearrangements with it. A rare (14 out of 1,557 nested tandem duplications individual cases (Extended Data Fig. 8). kataegis. However, there mustalternative be additional determinants kataegis as with rearrangemutationalin processes, although we cannot exclude the possibility that foci, 0.9%) form of kataegis, of colocalizing ARTICLE Received 3 Dec 2015 | Accepted 12 Jun 2016 | Published 13 Jul 2016 DOI: 10.1038/ncomms12222 OPEN A whole-genome sequence and transcriptome perspective on HER2-positive breast cancers Anthony Ferrari1, Anne Vincent-Salomon2, Xavier Pivot3, Anne-Sophie Sertier1, Emilie Thomas1, Laurie Tonon1, Sandrine Boyault4, Eskeatnaf Mulugeta5, Isabelle Treilleux6, Gaëtan MacGrogan7, Laurent Arnould8, Janice Kielbassa1, Vincent Le Texier1, Hélène Blanché9, Jean-Franc¸ois Deleuze9, Jocelyne Jacquemier10, Marie-Christine Mathieu11, Frédérique Penault-Llorca12, Frédéric Bibeau13, Odette Mariani14, Cécile Mannina15, Jean-Yves Pierga16, Olivier Trédan17, Thomas Bachelot17, Hervé Bonnefoi18, Gilles Romieu19, Pierre Fumoleau8, Suzette Delaloge11, Maria Rios20, Jean-Marc Ferrero21, Carole Tarpin22, Catherine Bouteille23, Fabien Calvo24, Ivo Glynne Gut25,26, Marta Gut25,26, Sancha Martin27, Serena Nik-Zainal27,28, NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12222 Michael R. Stratton27, Iris Pauporté29, Pierre Saintigny30,31,32, Daniel Birnbaum33, Alain Viari1,34 & Gilles Thomas1 a RNA group: PAM50: ER/PR: HER2: HER2 CNV.: Ploidy: B C D LumA LumB Her2 Basal Pos Neg NA 3+ 2+ / FISH+ Amplified 2 Gained >2 NA PAM50 ER PR HER2 HER2.CNV PLOIDY FGA [ 75, 100 ] [ 50, 75 ] LST Quartiles (%):University, Département de Lyon Cancer, Plateforme de bioinformatique ‘Gilles Thomas’ Centre Léon Bérard, 28 rue Laënnec, 69008 Lyon, France. 2 Institut Curie, PSL Research [ 25, 50 ] Pathologie, INSERM U934, 26 rue d’Ulm, 75248 Paris, France. 3 Centre Hospitalier Universitaire de Minjoz, UMR INSERM 1098, Boulevard A. Fleming, Besanc¸on 25000, France. 4 Plateforme de génomique des cancers, Centre Léon Bérard, 28 rue Laënnec, 69008 Lyon, France. 5 Institut Curie, UMR 3215 CNRS, Génétique et biologie du développement, Epigénèse et [ 0 , 25 ] développement des mammifères, U934 Inserm, 26 rue d’Ulm, 75248 Paris, France. 6 Centre Léon Bérard, Département de Pathologie, 28 rue Laënnec, 69008 Lyon, France. 7 Département de Biopathologie, Unité Inserm U916, Institut Bergonié, 229 cours de l’Argonne, 33076 Bordeaux, France. 8 Centre Georges-Franc¸ois Leclerc et CRB Ferdinand Cabanne, 1 rue du Professeur Marion, Inserm U866-UBFC, 21000 Dijon, France. 9 Centre d’Etude du Polymorphisme Humain (CEPH), Fondation Jean Dausset, 27 rue Juliette Dodu, 75010 Paris, France. 10 Institut PaoliCalmettes, Département de Pathologie, 232 Boulevard de Sainte-Marguerite, 13009 Marseille, France. 11 Institut Gustave Roussy, Comité de Pathologie Mammaire, 114 rue Edouard Vaillant, 94805 Villejuif, France. 12 Centre Jean Perrin, Département de Biopathologie, EA 4677 ERTICa, Université d’Auvergne, 58 rue Montalembert, 63000 Clermont-Ferrand, France. 13 Institut Régional du Cancer de Montpellier (ICM), Département de Pathologie, 208 Avenue des Apothicaires, 34298 Montpellier, France. 14 Institut Curie, PSL Research University, Service de Pathologie, Centre de Ressources Biologiques, BRIF BB-0033-00048, 26 rue d’Ulm, 75248 Paris, France. 15 Département de Pathologie, Institut Bergonié, 229 cours de l’Argonne, CS 61283, 33076 Bordeaux, France. 16 Institut Curie, PSL Research University, Département d’Oncologie Médicale, Université Paris Descartes, 26 rue d’Ulm, 75248 Paris, France. 17 Centre Léon Bérard, Département de Cancérologie Médicale, 28 rue Laënnec, 69008 Lyon, France. 18 Department of Medical Oncology, Institut Bergonié Unicancer, University of Bordeaux, INSERM U916, CIC1401, 229 cours de l’Argonne, CS 61283, 33076 Bordeaux, France. 19 Institut Régional du Cancer de Montpellier (ICM), Oncologie Sénologie, 208 Avenue des Apothicaires, 34298 Montpellier, France. 20 Centre Alexis Vautrin, Département d’Oncologie Médicale, 6 Avenue de Bourgogne, 54511 Vandoeuvre Les Nancy, France. 21 Centre Antoine Lacassagne, Département d’Oncologie Médicale, 33 Avenue de Valombrose, 06189 Nice, France. 22 Institut Paoli-Calmettes, Département d’Oncologie Médicale, 232Missense Boulevard de mutation Sainte-Marguerite, 13009 Marseille, France. 23 Clinique Mutualiste de Bellevue, Chirurgie Gynécologique et Mammaire, 3 rue le Verrier, 42100 Saint-Etienne, France. 24 Institut Gustave Roussy, Cancer Core 26 Universitat Pompeu Fabra, Europe, 39 rue Camille Desmoulins, Villejuif 94805, France. 25 CNAG-CRG, Centre for Genomic Regulation (CRG), C/Baldiri Reixac 4, 08028 Barcelona, Spain. Truncating mutation Plac¸a de la Mercè, 10, 08002 Barcelona, Spain. 27 Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. 28 East Anglian Medical Genetics Service, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 9NB, UK. 29 Institut National du Cancer, Département de Recherche Clinique, 52 Avenue Morizet, 92513 Boulogne-Billancourt, France. Amplification 30 INSERM U1052-CNRS 5286, Cancer Research Center of Lyon, F-69008 Lyon, France. 31 Université de Lyon, F-69622 Lyon, France. 32 Centre Léon Bérard, 28 rue Laënnec, 69008 Lyon, France. 33 Département d’Oncologie Moléculaire, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, INSERM, CNRS, Aix-Marseille Université, 232 boulevard de Sainte-Marguerite, 13009 Marseille, France. 34 Equipe Erable, INRIA Grenoble-Rhône-Alpes, 655 Avenue de l’Europe, 38330 Montbonnot-Saint Martin,Homozygous France. Correspondence and deletion requests for materials should be addressed to A.F. (email: [email protected]) or to A.V. (email: [email protected]). 1 Synergie b NATURE COMMUNICATIONS | 7:12222 | DOI: 10.1038/ncomms12222 | www.nature.com/naturecommunications RNA A 1 pLum mLum 0 10 20 30 40 50 SV17 TP53 PIK3CA KMT2C JAK2 KMT2D MAP2K4 GATA3 CDH1 NF1 ERBB2 CDK12 PPM1D RPS6KB1 TOP2A CCND1 KAT7 BCAS3 MYC ZNF703 RAD21 ZNF217 FGFR1 MCL1 MDM4 HMGA2 MDM2 GNAS PI001 PI046 PI049 PI053 PI059 PI073 PI078 PI085 PI088 PI089 PI099 PI017 PI020 PI038 PI061 PI074 PI084 PI093 PI036 PI042 PI005 PI003 PI009 PI014 PI015 PI019 PI023 PI027 PI044 PI048 PI051 PI055 PI062 PI079 PI068 PI013 PI052 PI069 PI045 PI012 PI011 PI071 PI006 PI098 PI002 PI024 PI056 PI064 PI096 PI097 PI091 PI035 PI063 PI076 PI010 PI041 PI057 PI066 PI082 PI090 PI094 PI022 PI034 PI092 HER2-positive breast cancer has long proven to be a clinically distinct class of breast cancers for which several targeted therapies are now available. However, resistance to the treatment associated with specific gene expressions or mutations has been observed, revealing the underlying diversity of these cancers. Therefore, understanding the full extent of the HER2-positive disease heterogeneity still remains challenging. Here we carry out an in-depth genomic characterization of 64 HER2-positive breast tumour genomes that exhibit four subgroups, based on the expression data, with distinctive genomic features in terms of somatic mutations, copy-number changes or structural variations. The results suggest that, despite being clinically defined by a specific gene amplification, HER2-positive tumours melt into the whole luminal–basal breast cancer spectrum rather than standing apart. The results also lead to a refined ERBB2 amplicon of 106 kb and show that several cases of amplifications are compatible with a breakage–fusion–bridge mechanism. ARTICLE ERBB2 amplifica-on mechanism Anthony Ferrari, Anne Vincent-­‐Salomon, Xavier Pivot et al Nature comm 2016 PI078 ! ! 19 18 17 16 Breakage-­‐Fusion-­‐Bridge 15 14 13 12 12 tumoral copy number 12 11 10 9 8 8 7 6 5 4 3 2 2 1 0 ? 3.60e+07 3.65e+07 3.70e+07 > 3.75e+07 3.80e+07 < > 3.85e+07 3.90e+07 position PI034 !! ! 25 24 23 22 21 20 18 19 18 16 17 from MaroZa et al. Breast Cancer Research 2012 tumoral copy number 16 14 15 14 14 12 13 12 10 11 10 8 9 8 7 6 5 4 2 3 2 1 0 ? 3.4e+07 3.6e+07 < < ? > < > 3.8e+07 position > 4.0e+07 4.2e+07 HER-­‐posi-ve breast cancer heterogeneity: a whole genome sequence and transcriptome perspec-ve Anthony Ferrari, Anne Vincent-­‐Salomon, Xavier Pivot et al Nature comm 2016 100 80 60 40 q24.3 q25.1 q25.2 q25.3 q24.3 q25.2 q25.1 q25.2 q25.3 q25.3 q24.2 q24.1 q23.3 q23.2 q23.1 q22 q21.33 q21.32 q21.31 q21.1 q21.2 q12 q11.2 q11.1 p11.1 p11.2 p12 p13.1 0 p13.2 0 p13.3 20 20 40 60 80 100 ERBB2 100 100 CDK12 KAT7 TOP2A 60 60 PPM1D SPOP BCAS3 40 40 20 q24.2 q25.1 q24.1 q23.1 q22 q23.2 q21.33 q22 q21.33 q21.32 q21.31 q21.32 q21.1 q21.2 q12 q21.31 q11.2 q21.2 q11.1 p11.1 q21.1 p11.2 q12 p12 q11.2 p13.1 0 p13.2 0 q23.3 q23.1 q24.1 q23.2 q24.2 q24.3 q23.3 DDX5 20 20 p13.1 p13.3 p11.2 q11.1 frequency 80 80 RPS6KB1 amplifications HER-­‐posi-ve breast cancer heterogeneity: a whole genome sequence and transcriptome perspec-ve Anthony Ferrari, Anne Vincent-­‐Salomon, Xavier Pivot et al Nature comm 2016 106 kb 100 0 IKZF3 GRB7 MIEN1 ERBB2 PGAP3 TCAP PNMT 20 STARD3 40 PPP1R1B 60 NEUROD2 80 RNA HER-­‐posi-ve breast cancer heterogeneity: a whole genome sequence and transcriptome perspec-ve PAM50 ER PR Anthony Ferrari, Anne Vincent-­‐Salomon, Xavier Pivot et al HER2 Nature comm 2016 HER2.CNV PLOIDY FGA LST pLum 0 10 20 30 40 50 10 20 30 40 50 PAM50 SV17 0 RNA mLum ER TP53 PIK3CA PR KMT2C HER2 JAK2 KMT2D HER2.CNV MAP2K4 GATA3 PLOIDY CDH1 FGA NF1 Truncating mutation! Amplification! Homozygous deletion! ERBB2 LST CDK12 pLum PPM1D RPS6KB1 mLum TOP2A SV17 CCND1 KAT7 TP53 BCAS3 PIK3CA MYC KMT2C ZNF703 JAK2 RAD21 KMT2D ZNF217 MAP2K4 FGFR1 GATA3 MCL1 CDH1 MDM4 NF1 HMGA2 ERBB2 MDM2 CDK12 GNAS PPM1D RPS6KB1 TOP2A CCND1 KAT7 BCAS3 MYC ZNF703 RAD21 ZNF217 FGFR1 PI001 PI046 PI049 PI053 PI059 PI073 PI078 PI085 PI088 PI089 PI099 PI017 PI020 PI038 PI061 PI074 PI084 PI093 PI036 PI042 PI005 PI003 PI009 PI014 PI015 PI019 PI023 PI027 PI044 PI048 PI051 PI055 PI062 PI079 PI068 PI013 PI052 PI069 PI045 PI012 PI011 PI071 PI006 PI098 PI002 PI024 PI056 PI064 PI096 PI097 PI091 PI035 PI063 PI076 PI010 PI041 PI057 PI066 PI082 PI090 PI094 PI022 PI034 PI092 Missense mutation! SIGNAL study : GWAS findings No associaGons between SNPs and HER2 status X Pivot et al Nature Journal Breast Cancer 2017