ARTICLE Received 13 Aug 2013 | Accepted 26 Feb 2014 | Published 27 Mar 2014 DOI: 10.1038/ncomms4518 Frequent mutations in chromatin-remodelling genes in pulmonary carcinoids Lynnette Fernandez-Cuesta1,*, Martin Peifer1,2,*, Xin Lu1, Ruping Sun3,LukaOzretic´4, Danila Seidel1,5, Thomas Zander1,6,7, Frauke Leenders1,5, Julie George1,ChristianMu¨ller1, Ilona Dahmen1, Berit Pinther1, Graziella Bosco1, Kathryn Konrad8, Janine Altmu¨ller8,9,10, Peter Nu¨rnberg2,8,9, Viktor Achter11,UlrichLang11,12, Peter M. Schneider13, Magdalena Bogus13,AlexSoltermann14, Odd Terje Brustugun15,16,ÅslaugHelland15,16, Steinar Solberg17, Marius Lund-Iversen18,SaschaAnse´n6,ErichStoelben19, Gavin M. Wright20, Prudence Russell21, Zoe Wainer20, Benjamin Solomon22, John K. Field23, Russell Hyde23, Michael P.A. Davies23, Lukas C. Heukamp4,7, Iver Petersen24,SvenPerner25, Christine M. Lovly26, Federico Cappuzzo27, William D. Travis28,Ju¨rgen Wolf5,6,7, Martin Vingron3, Elisabeth Brambilla29,StefanA.Haas3, Reinhard Buettner4,5,7 & Roman K. Thomas1,4,5 Pulmonary carcinoids are rare neuroendocrine tumours of the lung. The molecular alterations underlying the pathogenesis of these tumours have not been systematically studied so far. Here we perform gene copy number analysis (n ¼ 54), genome/exome (n ¼ 44) and transcriptome (n ¼ 69) sequencing of pulmonary carcinoids and observe frequent mutations in chromatin-remodelling genes. Covalent histone modifiers and subunits of the SWI/SNF complex are mutated in 40 and 22.2% of the cases, respec- tively, with MEN1, PSIP1 and ARID1A being recurrently affected. In contrast to small-cell lung cancer and large-cell neuroendocrine lung tumours, TP53 and RB1 mutations are rare events, suggesting that pulmonary carcinoids are not early progenitor lesions of the highly aggressive lung neuroendocrine tumours but arise through independent cellular mechanisms. These data also suggest that inactivation of chromatin-remodelling genes is sufficient to drive transformation in pulmonary carcinoids. 1 Department of Translational Genomics, Center of Integrated Oncology Cologne–Bonn, Medical Faculty, University of Cologne, 50924 Cologne, Germany. 2 Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany. 3 Computational Molecular Biology Group, Max Planck Institute for Molecular Genetics, D-14195 Berlin, Germany. 4 Department of Pathology, University Hospital Medical Center, University of Cologne, 50937 Cologne, Germany. 5 Laboratory of Translational Cancer Genomics, Center of Integrated Oncology Cologne—Bonn, University of Cologne, 50924 Cologne, Germany. 6 Department I of Internal Medicine, Center of Integrated Oncology Ko¨ln-Bonn, University of Cologne, 50924 Cologne, Germany. 7 Network Genomic Medicine, University Hospital Cologne, Center of Integrated Oncology Cologne Bonn, 50924 Cologne, Germany. 8 Cologne Center for Genomics (CCG), University of Cologne, 50931 Cologne, Germany. 9 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany. 10 Institute of Human Genetics, University of Cologne, Cologne 50931, Germany. 11 Computing Center, University of Cologne, 50931 Cologne, Germany. 12 Department of Informatics, University of Cologne, 50931 Cologne, Germany. 13 Institute of Legal Medicine, University of Cologne, 50823 Cologne, Germany. 14 Institute for Surgical Pathology, University Hospital Zurich, 8091 Zurich, Switzerland. 15 Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, N- 0424 Oslo, Norway. 16 Department of Oncology, Norwegian Radium Hospital, Oslo University Hospital, N-0310 Oslo, Norway. 17 Department of Thoracic Surgery, Rikshospitalet, Oslo University Hospital, N-0027 Oslo, Norway. 18 Department of pathology, Norwegian Radium Hospital, Oslo University Hospital, N-0310 Oslo, Norway. 19 Thoracic Surgery, Lungenklinik Merheim, Kliniken der Stadt Ko¨ln gGmbH, 51109 Cologne, Germany. 20 Department of Surgery, St Vincent’s Hospital, University of Melbourne, Melbourne, Victoria 3065, Australia. 21 Department of Pathology, St Vincent’s Hospital, Melbourne, Victoria 3065, Australia. 22 Department of Haematology and Medical Oncology, Peter MacCallum Cancer Centre, Melbourne, Victoria 3002, Australia. 23 Roy Castle Lung Cancer Research Programme, Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool Cancer Research Centre, Liverpool L3 9TA, UK. 24 Institute of Pathology, Jena University Hospital, Friedrich-Schiller-University, 07743 Jena, Germany. 25 Department of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, 53127 Bonn, Germany. 26 Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232, USA. 27 Department of Medical Oncology, Istituto Toscano Tumouri, 57100 Livorno, Italy. 28 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA. 29 Department of Pathology, CHU Grenoble INSERM U823, Institute Albert Bonniot 38043, CS10217 Grenoble, France. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to R.K.T., (email: roman.thomas@uni-Ko¨ln.de). NATURE COMMUNICATIONS | 5:3518 | DOI: 10.1038/ncomms4518 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4518 ulmonary carcinoids are neuroendocrine tumours that NonFermentable) complex are mutated in 40 and 22.2% of the account for about 2% of pulmonary neoplasms. On the cases, respectively. By contrast, mutations of TP53 and RB1 are Pbasis of the WHO classification of 2004, carcinoids can be only found in 2 out of 45 cases, suggesting that these genes are subdivided into typical or atypical, the latter ones being very rare not main drivers in pulmonary carcinoids. (about 0.2%)1. Most carcinoids can be cured by surgery; however, inoperable tumours are mostly insensitive to chemo- and radiation therapies1. Apart from few low-frequency alterations, Results such as mutations in MEN1 (ref. 1), comprehensive genome In total, we generated genome/exome sequencing data for 44 analyses of this tumour type have so far been lacking. independent tumour-normal pairs, and for most of them, also Here we conduct integrated genome analyses2 on data from RNAseq (n ¼ 39, 69 in total) and SNP 6.0 (n ¼ 29, 54 in total) chromosomal gene copy number of 54 tumours, genome and data (Supplementary Table 1). Although no significant focal exome sequencing of 29 and 15 tumour-normal pairs, copy number alterations were observed across the tumours respectively, as well as transcriptome sequencing of 69 tumours. analysed, we detected a copy number pattern compatible with Chromatin-remodelling is the most frequently mutated pathway chromothripsis3 in a stage-III atypical carcinoid of a former in pulmonary carcinoids; the genes MEN1, PSIP1 and ARID1A smoker (Fig. 1a; Supplementary Fig. 1). The intensely clustered were recurrently affected by mutations. Specifically, covalent genomic structural alterations found in this sample were histone modifiers and subunits of the SWI/SNF (SWich/Sucrose restricted to chromosomes 3, 12 and 13, and led to the a b 22 4 20 21 1 19 18 2 3 17 2 16 3 15 1 Copy number 14 0 13 4 12 5 004080120160 200 40 80 120 0 40 80 120 11 chr3 (Mbp) chr12 (Mbp) chr13 (Mbp) 10 6 Copy number Intra-chromosomal Inter-chromosomal 9 7 Raw copy number 8 segments chimeric transcript chimeric transcript c d 12 1.0 SQ 10 0.8 AD 8 SCLC G:C>A:T 0.6 A:T>G:C 6 C:G>A:T Muts/Mb Mutation rate 0.4 C:G>G:C 4 A:T>T:A A:T>C:G 0.2 2 PCA 0 0.0 SCLC Evol PCA Figure 1 | Genomic characterization of pulmonary carcinoids. (a) CIRCOS plot of the chromothripsis case. The outer ring shows chromosomes arranged end to end. Somatic copy number alterations (gains in red and losses in blue) detected by 6.0 SNP arrays are depicted in the inside ring. (b)Copy numbers and chimeric transcripts of affected chromosomes. Segmented copy number states (blue points) are shown together with raw copy number data averaged over 50 adjacent probes (grey points). To show the different levels of strength for the identified chimeric transcripts, all curves are scaled according to the sequencing coverage at the fusion point. (c) Mutation frequency detected by genome and exome sequencing in pulmonary carcinoids (PCA). Each blue dot represents the number of mutations (muts) per Mb in one pulmonary carcinoid sample. Average frequencies are also shown for adenocarcinomas (AD), squamous (SQ) and small-cell lung cancer (SCLC) based on previous studies2,4,5.(d) Comparison of context-independent transversion and transition rates (an overall strand symmetry is assumed) between rates derived from molecular evolution (evol)36, from a previous SCLC sequencing study2 and from the PCA genome and exome sequencing. All rates are scaled such that their overall sum is 1. 2 NATURE COMMUNICATIONS | 5:3518 | DOI: 10.1038/ncomms4518 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4518 ARTICLE expression of several chimeric transcripts (Fig. 1b; Supplementary MEN1 is a molecular adaptor that physically links MLL with the Table 2). Some of these chimeric transcripts affected genes chromatin-associated protein PSIP1, an interaction that is involved in chromatin-remodelling processes, including out-of- required for MLL/MEN1-dependent functions7. MEN1 also acts frame fusion