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. 2015 Aug 6;524(7563):47-53.
doi: 10.1038/nature14664. Epub 2015 Jul 13.

Comprehensive genomic profiles of small cell lung cancer

Julie George  1 Jing Shan Lim  2 Se Jin Jang  3 Yupeng Cun  1 Luka Ozretić  4 Gu Kong  5 Frauke Leenders  1 Xin Lu  1 Lynnette Fernández-Cuesta  1 Graziella Bosco  1 Christian Müller  1 Ilona Dahmen  1 Nadine S Jahchan  2 Kwon-Sik Park  2 Dian Yang  2 Anthony N Karnezis  6 Dedeepya Vaka  2 Angela Torres  2 Maia Segura Wang  7 Jan O Korbel  7 Roopika Menon  8 Sung-Min Chun  3 Deokhoon Kim  9 Matt Wilkerson  10 Neil Hayes  11 David Engelmann  12 Brigitte Pützer  12 Marc Bos  1 Sebastian Michels  13 Ignacija Vlasic  14 Danila Seidel  1 Berit Pinther  1 Philipp Schaub  1 Christian Becker  15 Janine Altmüller  16 Jun Yokota  17 Takashi Kohno  18 Reika Iwakawa  18 Koji Tsuta  19 Masayuki Noguchi  20 Thomas Muley  21 Hans Hoffmann  22 Philipp A Schnabel  23 Iver Petersen  24 Yuan Chen  24 Alex Soltermann  25 Verena Tischler  25 Chang-min Choi  26 Yong-Hee Kim  27 Pierre P Massion  28 Yong Zou  28 Dragana Jovanovic  29 Milica Kontic  29 Gavin M Wright  30 Prudence A Russell  31 Benjamin Solomon  32 Ina Koch  33 Michael Lindner  33 Lucia A Muscarella  34 Annamaria la Torre  34 John K Field  35 Marko Jakopovic  36 Jelena Knezevic  37 Esmeralda Castaños-Vélez  38 Luca Roz  39 Ugo Pastorino  40 Odd-Terje Brustugun  41 Marius Lund-Iversen  42 Erik Thunnissen  43 Jens Köhler  44 Martin Schuler  44 Johan Botling  45 Martin Sandelin  45 Montserrat Sanchez-Cespedes  46 Helga B Salvesen  47 Viktor Achter  48 Ulrich Lang  49 Magdalena Bogus  50 Peter M Schneider  50 Thomas Zander  51 Sascha Ansén  13 Michael Hallek  52 Jürgen Wolf  13 Martin Vingron  53 Yasushi Yatabe  54 William D Travis  55 Peter Nürnberg  56 Christian Reinhardt  14 Sven Perner  9 Lukas Heukamp  4 Reinhard Büttner  4 Stefan A Haas  53 Elisabeth Brambilla  57 Martin Peifer  58 Julien Sage  2 Roman K Thomas  59
Affiliations

Comprehensive genomic profiles of small cell lung cancer

Julie George et al. Nature. .

Abstract

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

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Figures

Extended Data Figure 1
Extended Data Figure 1. Genomic analyses in SCLC tumours
a, Schematic detailing the genomic study and number of samples as well as various steps of analyses for the identification of candidate genes in SCLC. b, Illustration of the number of samples analysed in this study.
Extended Data Figure 2
Extended Data Figure 2. Clinical molecular-correlation analyses
a, Survival analysis of SCLC patients based on clinical stage and treatment options (surgery and/or chemotherapy). Statistical significance was determined by log-rank test. b, Analyses of clinical stage and smoking status and the respective effect on number and type of mutations, as well as mutational subclonality in tumours. Statistical significance was determined by Kruskal–Wallis analysis.
Extended Data Figure 3
Extended Data Figure 3. Genomic characterization of SCLC tumours
a, Purity and ploidy determined in SCLC tumours by whole-genome sequencing presented as dot density plots showing median and the interquartile range (IQR) b, Subclonal architecture of SCLC in comparison to lung adenocarcinoma (AD). Whole-genome sequencing data of SCLC and of adenocarcinoma (n = 15) was analysed for the presence of subclonal populations using clustering of the derived cancer cell fraction (CCF) of all single nucleotide mutations. To compare the emerging subclonal structure, we derived a subclonality score that takes into account the CCF of each subpopulation as well as its mutational burden (see Methods). In order to prevent the low sequencing coverage (35 × for SCLC and 63 × for AD) from causing a systematic underrepresentation of the subclonal diversity in the mutation calls, we computed the contribution of a single read to the CCF on genome-wide average. After systematically determining a threshold within the average increase of CCF per read values (see Methods for details), we determined the group of samples for which a reliable estimation of the subclonality score is not possible (grey area). The subclonality scores of the remaining SCLC cases were then compared to those of the adenocarcinoma cases (P = 0.000232; Mann–Whitney test). c, Schematic representation of candidate genes with significant clustering of mutations in respective protein domains. Somatic mutations and genomic translocations are mapped to the respective protein regions. Hotspot mutations are highlighted in red. d, e, Genomic alterations in the RB1 family proteins p107 (RBL1) and p130 (RBL2) (d), and in KIT and PIK3CA (e). Somatic mutations in therapeutic target genes are listed and mapped to the protein domains of KIT and PIK3CA. Mutations with potential therapeutic implications are highlighted in red.
Extended Data Figure 4
Extended Data Figure 4. Clinical molecular-correlations of significantly mutated genes
a, Survival analysis of SCLC patients based on the status of CREBBP/EP300, TP73 or NOTCH alterations. Statistical significance was determined by log-rank test. b, Analysis of CREBBP/EP300, TP73 and NOTCH alterations and their effect on clinical and genetic parameters. Statistical significance was analysed by multinomial logistic regression.
Extended Data Figure 5
Extended Data Figure 5. Significant somatic copy number alterations in SCLC
a, Deletions of the chromosomal arm 3p point to the 3p14 (FHIT) and 3p12 (ROBO1) locus. b, Expression analyses of genes encoded on the 3p14.3–3p14.2 and 3p12.2–3p12.2 locus. Histogram displaying the expression of samples with focal deletions (blue) and samples without any copy number alterations (white). Mean and standard error of the mean is plotted for each gene in each group. Significant differences were determined by Mann–Whitney test; *P < 0.05; **P < 0.01. c, d, Focal deletions of the CDKN2A (c) and focal amplifications of IRS2 (d) were found on chromosome 9 and 13, respectively. The copy number (CN) states were computed from SNP array (SNP 6.0) and from whole-genome sequencing (WGS) data. The samples are sorted according to their amplitude of deletions or amplifications. e, Amplifications of IRS2 were determined by FISH analysis. IRS amplifications were quantified based on the ratio of red signals (IRS2-specific probe) to green signals (centromere probe for chromosome 13). Lymphocyte spreads and SCLC tumours without detectable IRS2 amplifications served as negative controls. Scale bar, 100 µm.
Extended Data Figure 6
Extended Data Figure 6. TP53 and RB1 alterations in SCLC
a, Distribution of somatic mutations in TP53 and RB1 according to the colour panel provided. b, c, Complex genomic rearrangements in RB1 showing homozygous deletions of exon 1 (b) or inversions within the RB1 gene (c). d, e, Annotated silent or missense mutations in RB1 occur at intron-exon junctions resulting in alternative splicing, intron retention (d) or exon skipping events (e). The coverage at the respective exon junctions is quantified as RPKM values. Sample S02194 is not holding any mutations at intron–exon junctions and is displayed as an example for unaltered splicing of RB1.
Extended Data Figure 7
Extended Data Figure 7. Chromothripsis in human SCLC
a, Circos plot of the chromothripsis sample S02353 showing intra- and interchromosomal rearrangements between chromosome 3 and 11. The integral copy number state (iCN) is plotted as a heatmap and assigned to the respective chromosomal regions. The chromosomal context of CCND1 (on chromosome 11) is highlighted. b, Circos plots displaying fusion transcripts identified in the SCLC chromothripsis cases (Supplementary Table 12) are represented as blue (S02297) or red (S02353) lines for genes located on chromosome 3 and 11. c, Immunohistochemistry staining for p53, p14 (ARF) and p16 on FFPE material of the chromothripsis sample S02297. Original magnification, ×400.
Extended Data Figure 8
Extended Data Figure 8. Recurrent genomic translocations in SCLC
a, Recurrent genomic translocations (n = 14) affecting chromosome 22 are illustrated as a Circos plot highlighting the respective rearrangements as red connecting lines. b, Breakpoints in chromosome 22 map to intron 1 of TTC28 and cluster downstream of the LINE1 (L1Hs) retrotransposon. Each arrow indicates the sample and the respective chromosomal position the segment translocates to. c, Schematic representation of the TP73 locus (hg19) describing complex intrachromosomal rearrangements of TP73 identified for S02397 and S02243. Recurrent somatic mutations identified in Fig. 1a are mapped to the respective exons. d, Validation of somatic TP73 translocations. Genomic regions involved in the TP73 rearrangements were amplified in matched normal (N) and tumour (T) samples. The expected band size is indicated in brackets. The respective PCR products were subjected to Sanger sequencing to confirm the genomic breakpoint. e, Copy-number state of the TP73 gene in samples involved in genomic translocations.
Extended Data Figure 9
Extended Data Figure 9. Transcriptome profile of human SCLC tumours
a, Unsupervised hierarchical clustering of transcriptome sequencing data of 69 SCLC specimens as described in Fig. 4a. Each sample is annotated for the genomic alterations described in Fig. 1. Black filled boxes describe the presence of a genomic event. b, Expression values of CHGA, GRP, ASCL1 and DLK1 (FPKM) are represented as dot density plots for the subgroups identified in (a). Red lines highlight the median value for each group. c, Expression values of the neuroendocrine markers SYP (synaptophysin) and NCAM1 (CD56) plotted as scatter plots for all SCLC samples. Green lines indicate thresholds for no expression (FPKM<1).
Extended Data Figure 10
Extended Data Figure 10. Notch is a tumour suppressor in SCLC regulating neuroendocrine differentiation
a, Somatic mutations identified in NOTCH3 and NOTCH4 are mapped to the protein domains. Damaging mutations are highlighted in red. Mutations found in murine SCLC tumours are highlighted in blue. b, Quantification of tumour lesions and per cent tumour area to lung in TKO (n = 5) and TKO;N1ICD (n = 4) mice 3 months after Ad-Cre instillation. Statistical significance was determined by two-tailed unpaired Student’s t-test. c, Representative immunohistochemistry for GFP or tdTomato in lungs from TKO;Rosa26mT/mG mice approximately 6 months after tumour induction. Left scale bar, 500 µm; right and middle: scale bar, 50 µm. d, Representative immunostaining for Notch2 in lungs from TKO;Rosa26N2ICD mice approximately 6 months after tumour induction. Left scale bar, 500 µm; right scale bar, 50 µm. e, Quantification of the per cent recombination at the Rosa26 locus in TKO;Rosa26mT/mG (n = 6) and TKO;Rosa26N2ICD mice (n = 10; two-tailed unpaired Student’s t-test). f, Cell viability assay of the human SCLC cell line NJH29 transfected with a N1ICD (Notch1) expression plasmid or empty vector control (Ctrl) (3 independent biological replicas with 3 technical replicas each). Fold growth was normalized to day 0; representative images were taken on day 6. Scale bar, 50 µm. g, Immunohistochemistry staining in FFPE embedded tissues of TKO and TKO;N2ICD mice. Scale bar, 50 µm. h, Quantitative RT–PCR validation of Notch1 induction and the expression of common Notch target genes after N1ICD transfection in murine SCLC cells (three biological replicas; two-tailed paired Student’s t-test). i, Mouse SCLC cells transfected with control or N1ICD (Notch1) were analysed 48 h later by gene expression microarrays. Gene Set Enrichment Analysis (GSEA) was performed on these data; selected significant gene sets are displayed. j, k, EdU analysis of mouse (j) and human (k) SCLC cells (three independent biological replicas with three technical replicas each; two-tailed paired Student’s t-test). *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as mean ± s.d.
Figure 1
Figure 1. Genomic alterations in small cell lung cancer
a, Tumour samples are arranged from left to right. Alterations of SCLC candidate genes are annotated for each sample according to the colour panel below the image. The somatic mutation frequencies for each candidate gene are plotted on the right panel. Mutation rates and type of base-pair substitution are displayed in the top and bottom panel, respectively. Significant candidate genes are highlighted in bold (*corrected q-values < 0.05, †P < 0.05, ‡P < 0.01). The respective level of significance is displayed as a heatmap on the right panel. Genes that are also mutated in murine SCLC tumours are denoted with a § symbol. Mutated cancer census genes of therapeutic relevance are denoted with a + symbol. b, Somatic copy number alterations determined for 142 human SCLC tumours by single nucleotide polymorphism (SNP) arrays. Significant amplifications (red) and deletions (blue) were determined for the chromosomal regions and are plotted as q-values (significance < 0.05).
Figure 2
Figure 2. Universal bi-allelic inactivation of TP53 and RB1 in human SCLC
a, Alterations of TP53 and RB1 were determined based on whole-genome sequencing data of 108 SCLC cases. Samples are plotted from left to right. Alleles A and B are represented for each case and colour-coded according to the somatic alteration. The integral copy number (iCN) state of each allele is plotted; hemizygous losses are annotated as loss of heterozygosity (LOH), copy-neutral LOH or LOH at higher ploidy. Samples retaining allele A and B show alterations on both alleles (bi-allelic alterations). b, Circos plot of case S02297 showing intra- and interchromosomal translocations between chromosome 3 and 11. The copy number state of the respective chromosomal regions (iCN) is plotted as a heatmap. The genomic context of CCND1 (on chromosome 11) is highlighted. c, Significantly differentially expressed genes encoded on chromosome 11 are analysed in both chromothripsis cases in comparison to all other tumours. Positive and negative z-scores show upregulation and downregulation of genes, respectively (P < 0.05; *q-value < 0.05). d, Distribution of CCND1 expression over 81 SCLC samples. Chromothripsis cases are highlighted in red. e, Haematoxylin and eosin (H&E) and immunohistochemistry staining for cyclin D1 and Rb1 for sample S02297. Original magnification, ×400.
Figure 3
Figure 3. Recurrent rearrangements generating oncogenic variants of TP73
a, Genomic breakpoints identified by whole-genome sequencing were mapped to their chromosomal locations. Recurrent breakpoints (n > 6 samples) are highlighted in colours. b, Schematic representation of the TP73 locus (hg19) illustrating intragenic translocations. Coding and non-coding regions of the annotated exons are shown as black and white boxes, respectively. c, Schematic representation of exons encoding p73, p73Δex2, p73Δex2/3 and p73Δex10. d, Exon skipping events were assessed in the transcriptome data of samples with genomic translocations resulting in p73Δex2, p73Δex2/3 and p73Δex10 transcript variants. S02139 served as a reference sample without TP73 alterations. The expression of uncommon exon combinations is highlighted in red.
Figure 4
Figure 4. Notch is a tumour suppressor and a key regulator of neuroendocrine differentiation in SCLC
a, Unsupervised expression analysis of human SCLC tumours. Tumour samples are arranged in columns and grouped by the expression of differentially expressed genes (rows). Expression values are represented as a heatmap; yellow and blue indicate high and low expression, respectively. b, Schematic representation of NOTCH1 and NOTCH2. Somatic mutations are mapped to the respective protein domains. Damaging and missense mutations are highlighted in red and black, respectively. c, Representative H&E images of lungs from Trp53;Rb1;Rbl2 triple-knockout (TKO) or TKO;N2ICD (Notch2) mice collected 3 months after Ad-Cre instillation. Scale bar, 1 mm. Tumours were quantified for each genotype (n = 8). Statistical significance was determined by two-tailed unpaired Student’s t-test. d, Survival analysis of TKO (n = 7, median survival = 210 days) and TKO;N2ICD (n = 8, median survival = 274 days) mice. Statistical significance was determined by log-rank test. e, Cell viability assay of the murine SCLC cell line KP1 transfected with a N1ICD (Notch1) expression plasmid or empty vector control (Ctrl) (3 independent biological replicas with 3 technical replicas each). Fold growth is normalized to day 0; representative images were taken on day 8. Scale bar, 50 µm. Statistical significance was determined by two-tailed paired Student’s t-test. f, Mouse SCLC cells were transfected with control or N1ICD and analysed 48 h after transfection by gene expression microarrays. The heatmap describes differentially expressed genes in control or N1ICD-transfected cells (n = 3, each); red and green indicate high and low expression, respectively. *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as mean ± s.d.
Figure 5
Figure 5. Signalling pathways recurrently affected in SCLC
Red and blue boxes denote genes with activating and inactivating alterations, respectively. Deep blue boxes highlight the bi-allelic inactivation of TP53 and RB1. Genes found expressed at high levels are shown in red font.

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