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LETTER doi:10.1038/nature09515 Distant metastasis occurs late during the genetic evolution of pancreatic cancer Shinichi Yachida1*, Siaˆn Jones2*, Ivana Bozic3, Tibor Antal3,4, Rebecca Leary2, Baojin Fu1, Mihoko Kamiyama1, Ralph H. Hruban1,5, James R. Eshleman1, Martin A. Nowak3, Victor E. Velculescu2, Kenneth W. Kinzler2, Bert Vogelstein2 & Christine A. Iacobuzio-Donahue1,5,6 Metastasis, the dissemination and growth of neoplastic cells in an were present in the primary pancreatic tumours from which the meta- organ distinct from that in which they originated1,2, is the most stases arose. A small number of these samples of interest were cell lines common cause of death in cancer patients. This is particularly true or xenografts, similar to the index lesions, whereas the majority were for pancreatic cancers, where most patients are diagnosed with fresh-frozen tissues that contained admixed neoplastic, stromal, metastatic disease and few show a sustained response to chemo- inflammatory, endothelial and normal epithelial cells (Fig. 1a). Each therapy or radiation therapy3. Whether the dismal prognosis of tissue sample was therefore microdissected to minimize contaminat- patients with pancreatic cancer compared to patients with other ing non-neoplastic elements before purifying DNA. types of cancer is a result of late diagnosis or early dissemination of Two categories of mutations were identified (Fig. 1b). The first and disease to distant organs is not known. Here we rely on data gen- largest category corresponded to those mutations present in all samples erated by sequencing the genomes of seven pancreatic cancer meta- from a given patient (‘founder’ mutations, mean of 64%, range 48–83% stases to evaluate the clonal relationships among primary and of all mutations per patient; Fig. 1b, example in Supplementary Fig. 2a). metastatic cancers. We find that clonal populations that give rise These data indicate that the majority of somatically acquired mutations to distant metastases are represented within the primary carcin- present in pancreatic cancers occur before the development of meta- oma, but these clones are genetically evolved from the original static lesions. All other mutations were characterized as ‘progressor’ parental, non-metastatic clone. Thus, genetic heterogeneity of mutations (mean of 36%, range 17–52% of all mutations per patient; metastases reflects that within the primary carcinoma. A quanti- tative analysis of the timing of the genetic evolution of pancreatic cancer was performed, indicating at least a decade between the a Pancreas Peritoneum Liver Lung occurrence of the initiating mutation and the birth of the parental, non-metastatic founder cell. At least five more years are required for the acquisition of metastatic ability and patients die an average of two years thereafter. These data provide novel insights into the genetic features underlying pancreatic cancer progression and define a broad time window of opportunity for early detection to prevent deaths from metastatic disease. b 90 We performed rapid autopsies of seven individuals with end stage Progressor pancreatic cancer (Supplementary Table 1). In all patients, metastatic 80 Founder deposits were present within two or more anatomic sites in each 70 patient, most often the liver, lung and peritoneum, as is typical for this form of neoplasia4. 60 Low passage cell lines (six patients) or first passage xenografts (one 50 patient) were created from one of the metastases present at each 40 patient’s autopsy. These samples comprised seven of the 24 pancreatic cancers which previously underwent whole exome sequencing and mutations Total 30 copy number analysis, as described in a mutational survey of the 20 5 pancreatic cancer genome . In this earlier study, a total of 426 somatic 10 mutations in 388 different genes were identified among 220,884,033 0 base pairs (bp) sequenced in the seven index metastatic lesions, cor- Pa01 Pa02 Pa03 Pa04 Pa05 Pa07 Pa08 responding to an average of 61 mutations per index metastatic lesion (range 41–77). In all samples, the vast majority of mutations were Figure 1 | Summary of somatic mutations in metastatic pancreatic cancers. represented by missense or silent single base substitutions (Sup- a, Histopathology of primary infiltrating pancreatic cancer and metastatic pancreatic cancer to the peritoneum, liver and lung. In addition to infiltrating plementary Fig. 1 and Supplementary Table 2). cancer cells in each lesion (arrows), non-neoplastic cell types are abundant. For each of the somatic mutations identified in the seven index b, Total mutations representing parental clones (founder mutations), and metastasis lesions, we determined whether the same somatic mutation clonal evolution (progressor mutations) within the primary carcinoma based was present in anatomically distinct metastases harvested at autopsy on comparative lesion sequencing. Mutations common to all samples analysed from the same patients. We also determined whether these mutations were the most common category identified. 1Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA. 2The Ludwig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical Institute at The Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland 21231, USA. 3Program for Evolutionary Dynamics, Department of Mathematics, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138, USA. 4School of Mathematics, University of Edinburgh, Edinburgh EH9 3JZ, UK. 5Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA. 6Department of Surgery, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA. *These authors contributed equally to this work. 1114 | NATURE | VOL 467 | 28 OCTOBER 2010 ©2010 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH Fig. 1b, example in Supplementary Fig. 2b). These mutations were upon which were superimposed an accumulation of progressor muta- present in one or more of the metastases examined, including the index tions associated with clonal evolution and metastasis. metastasis, but not the parental clone. Evolutionary maps were constructed for each patient’s carcinoma These mutation types were used to classify the lesions that contained based on the patterns of somatic mutation and allelic losses and the them into parental clones (containing only founder mutations) and locations of individual metastatic deposits (Fig. 2 and Supplementary subclones (containing both founder and progressor mutations). By Figs 3–8). These maps showed that, despite the presence of numerous definition, there could be only one parental clone in a patient, although founder mutations within the parental clones, the cells giving rise to there could be many different subclones. Parental clones tended to the metastatic lesions had a large number of progressor mutations. For contain more deleterious mutations (nonsense, splice site or frameshift example, in Pa01 the parental clone contained 49 founder mutations, mutations) than subclones (12.6% of the mutations in the parental yet a clonal expansion marked by the presence of mutations in six clones versus 8.1% of the mutations in subclones, Supplementary additional genes was present in the lung and peritoneal metastases Table 2). The parental clones had already accumulated mutations in (Supplementary Fig. 3). Moreover, 22 more mutations were found in all driver genes (KRAS, TP53 and SMAD4) previously shown to drive the liver metastasis. Note that all mutations in the metastatic lesions pancreatic tumorigenesis6. Through combined analysis of high-density were clonal, that is, present in the great majority if not all neoplastic single nucleotide polymorphism (SNP) chip data on the index lesion cells of the metastasis, as assessed by Sanger sequencing. Thus, these (Supplementary Table 3) plus the sequencing data on all lesions mutations were present in the cell that clonally expanded to become (Supplementary Table 2) we found that the vast majority of homo- the metastasis. Similarly, large numbers of progressor mutations were zygous mutations (51 mutations, representing 89% of all homozygous generally observed in the metastases from each of the seven cases mutations) in the index lesion were already present in the parental examined (Fig. 2 and Supplementary Figs 3–8). clones. Homozygous mutations are characteristic of tumour suppressor To distinguish between the possibilities that clonal evolution genes such as SMAD4 and CDKN2A and often occur in association with occurred inside the primary cancer versus within secondary sites, we chromosomal instability7. In sum, the parental clones harboured the sectioned the primary tumours from two patients into numerous, three- majority of deleterious genetic alterations and chromosomal instability, dimensionally organized pieces (Fig. 2a, b) and examined the DNA Slice number Slice 1 Slice 2 Slice 3 a 15432 b Y U B W T R V S Q G S D F A C E 3 cm RL Slice 4 I Slice 5 K Superior X P O J Right Left 5 mm NL Pancreatic tail Inferior H I M Necrosis Normal Parental c Pa08 C13orf22 Parental clone GPC2 GRM8 AKAP12 HPCAL1 ODZ4 C1RL MLL2 CCNB3 ATP2B3 ITPR1 OR4A16 B3GALT1 NEB CNGB3 PALMD BCL2A1 JPH4 OR5J2 LRRTM4 NP_001074311.1 CTNND2 RAD9B U CCNYL3 KAL1 OR8H1 SESN2 DICER1 LOC167127 MRGX1 NCL CGN C20orf26 P SYNE1