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.

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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 CMYA5 KIAA0082 SH3GL3 PNPLA1 FAT4 SLC25A26 EHMT1 V RDH8 OVCH1 SLC2A3 NLGN1 CNTNAP4 KIAA1957 SMAD4 O Q S T Y DNAH8 KRAS TBX6 W DOCK2 KRTAP11-1 TMEM132B R EML1 LGR6 TP53 X FLJ10324 MTR TTN FLJ21986 NEO1 TTN* GABRA1 NOD3

Primary carcinoma

e f a b c d Peritoneum Liver Liver Pa08 Lung (index Metastases lesion)

Figure 2 | Geographic mapping of metastatic clones within the primary circles. These clones are both geographically and genetically distinct from carcinoma and proposed clonal evolution of Pa08. a, Illustration of the clones giving rise to peritoneal metastases in this same patient, indicated in pancreatic specimen removed from Pa08 at rapid autopsy, and the planes of green. c, Proposed clonal evolution based on the sequencing data. In this model, sectioning of the specimen. b, Mapping of the parental clone and subclones after development of the parental clone, ongoing clonal evolution continues identified by comparative lesion sequencing within serial sections of the within the primary carcinoma (yellow rectangle), and these subclones seed infiltrating pancreatic carcinoma. Metastatic subclones giving rise to liver and metastases in distant sites. *Two mutations were found in the TTN gene. lung metastases are non-randomly located within slice 3, indicated by blue

28 OCTOBER 2010 | VOL 467 | NATURE | 1115 ©2010 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER from each piece for each of the founder and progressor mutations. In founder cell of the tumour. To estimate the timing, we first used Patient Pa08, there were three progressor mutations present in two Ki-67 labelling to determine the proliferation rate of seven samples independent peritoneal metastases (defining one subclone) and 23, of normal duct epithelium from surgically resected pancreata of indi- 25 or 27 additional progressor mutations present in liver and lung viduals without pancreatic cancer as well as of each index metastasis. metastases (defining three additional subclones; Fig. 2c). Through the Ki-67-positive nuclei constituted an average of 0.4% of normal ductal analysis of distinct regions of the primary tumour, it was clear that cells, whereas an average of 16.3% of cancer cells within the index subclones giving rise to each of these metastases were present in the metastasis lesions were Ki-67-positive, consistent with prior esti- primary tumour. Moreover, these subclones were not small; from the mates9,10 (Supplementary Table 4). Based on these data plus that from size of the pieces (Fig. 2a) and the amounts of DNA recovered, each sequencing of the index lesions, we derived estimates for three critical subclone must have contained in excess of 100 million cells. In addition, times in tumour evolution: T1, the time between tumour initiation and more than four different subclones, each containing a similarly large the birth of the cell giving rise to the parental clone; T2, the subsequent number of cells, could be identified through the analysis of other pieces time required for the birth of the cell that gave rise to the index of the same tumour. These subclones could be put into an ordered metastasis; and T3, the time between the dissemination of this cell hierarchy establishing an evolutionary path for tumour progression and the patients’ death (Fig. 3). In other words, there is a time point, (Fig. 2c). Analysis of multiple primary tumour pieces and metastatic t0, when the tumour was initiated, and a time point t1 when a cell is lesions from patient Pa04 revealed a similar clonal evolution, with born that has all mutations that exist in the parental clone. Similarly, distinct, large subclones within the primary tumours giving rise to the there is a time point in tumour evolution, t2, when a cell is born that has various metastases (Supplementary Fig. 8). all the mutations that exist in the index metastasis. T1 is given by t1 2 t0 To clarify further clonal evolution within the primary site, we and T2 is given by t2 2 t1. If we denote t3 as the time of patient’s death, attempted to correlate the mutation signatures representing the sub- then T3 5 t3 2 t2. clones of Pa08 (Fig. 2c) with the geographic location of the pieces of the Using the mathematical model described in the Methods, we were primary tumour used to define them (Fig. 2a, b). Samples representative able to conservatively estimate an average of 11.7 years from the ini- of the parental clone were located throughout the primary carcinoma. tiation of tumorigenesis until the birth of the cell giving rise to the By contrast, samples representing subclones were non-randomly parental clone, an average of 6.8 years from then until the birth of the located in proximity to each other, within which the subclones speci- cell giving rise to the index lesion, and an average of 2.7 years from then fically giving rise to peritoneal versus distant metastases were seen. until the patients’ death (see Supplementary Discussion and Sup- Thus, we conclude that the genetic heterogeneity of metastases reflects plementary Table 5). heterogeneity already existing within the primary carcinoma, and that We show, for the first time, that primary pancreatic cancers contain a the primary carcinoma is a mixture of numerous subclones, each of mix of geographically distinct subclones, each containing large numbers which has independently expanded to constitute a large number of cells. (hundreds of millions) of cells that are present within the primary This data set could also be used to infer the timing of the develop- tumour years before the metastases become clinically evident. The fea- ment of the various stages of pancreatic tumour progression8. We tures of these metastatic subclones that promote metastasis formation assume that the tumour is initiated by a genetic event that confers a have yet to be discerned, because no consistent genetic signature of selective growth advantage to the cell that goes on to become the metastatic subclones could be identified. We did identify several genes

Normal duct = Mutation(s) & epithelial cell clonal expansion = Dissemination to t Initiated 0 tumour cell distant organs

T1 (average of 11.7 ± 3.1 years)

Parental clone t1

T2 (average of 6.8 ± 3.4 years) Subclones with metastatic capacity

t2

T3 (average of 2.7 ± 1.2 years) Lung Liver Liver t3

Index lesion

Figure 3 | Schema of the genetic evolution of pancreatic cancer. the cell that will give rise to the index lesion will appear (end of T2 and Tumorigenesis begins with an initiating mutation in a normal cell that confers a beginning of T3). Unfortunately, most patients are not diagnosed until well into selective growth advantage. Successive waves of clonal expansion occur in time interval T3 when cells of these metastatic subclones have already escaped association with the acquisition of additional mutations, corresponding to the the pancreas and started to grow within distant organs. The average time for progression model of pancreatic intraepithelial neoplasia (PanIN) and time T1. intervals T1, T2 and T3 for all seven patients is indicated in the parentheses at left One founder cell within a PanIN lesion will seed the parental clone and hence (see also Supplementary Table 6). initiate an infiltrating carcinoma (end of T1 and beginning of T2). Eventually,

1116 | NATURE | VOL 467 | 28 OCTOBER 2010 ©2010 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH that were mutated in one or more of the index metastatic lesions from 3. Stathis, A. & Moore, M. J. Advanced pancreatic carcinoma: current treatment and future challenges. Nature Rev. Clin. Oncol. 7, 163–172 (2010). these seven patients with Stage IV disease, but not in the primary 4. Yachida, S. & Iacobuzio-Donahue, C. A. The pathology and genetics of metastatic pancreatic index lesions from 17 patients with Stage II disease (Sup- pancreatic cancer. Arch. Pathol. Lab. Med. 133, 413–422 (2009). plementary Table 2). These genes include those that may have a role 5. Jones, S. et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321, 1801–1806 (2008). in invasive or metastatic ability through heterotypic cell adhesion 6. Maitra, A. & Hruban, R. H. pancreatic cancer. Annu. Rev. Pathol. 3, 157–188 (2008). (CNTN5), motility (DOCK2), proteolysis (MEP1A) and tyrosine phos- 7. Lengauer, C., Kinzler, K. W. & Vogelstein, B. Genetic instabilities in human cancers. phorylation (LMTK2). However, these mutations were not metastasis- Nature 396, 643–649 (1998). specific per se as all but one were present in the matched primary 8. Jones, S. et al. Comparative lesion sequencing provides insights into tumor evolution. Proc. Natl Acad. Sci. USA 105, 4283–4288 (2008). carcinoma of those same seven patients, and there is no evidence that 9. Terada, T. et al. Cell proliferative activity in intraductal papillary-mucinous the mutations we observed endowed these genes with metastagenic neoplasms and invasive ductal adenocarcinomas of the pancreas: an activity. These data also do not reveal the selective pressures within immunohistochemical study. Arch. Pathol. Lab. Med. 122, 42–46 (1998). 10. Hisa, T. et al. Growth process of small pancreatic carcinoma: a case report with the primary carcinoma that led to the formation of progressor muta- imaging observation for 22 months. World J. Gastroenterol. 14, 1958–1960 tions. In light of recent findings indicating that pancreatic cancers are (2008). poorly vascularized11, one possibility is that intratumoural hypoxia cre- 11. Olive, K. P. et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 324, 1457–1461 ates a fertile microenvironment for the formation of additional muta- (2009). tions beyond that of the parental clone. 12. Sidransky, D. Emerging molecular markers of cancer. Nature Rev. Cancer 2, One of the major implications of these data is their implication for 210–219 (2002). 13. Embuscado, E. E. et al. Immortalizing the complexity of cancer metastasis: genetic screening to prevent pancreatic cancer deaths. Quantitative analysis features of lethal metastatic pancreatic cancer obtained from rapid autopsy. indicated a large window of opportunity for diagnosis while the disease Cancer Biol. Ther. 4, 548–554 (2005). was still in the curative stage—at least a decade. Our model also predicts 14. Fu, B. et al. Evaluation of GATA-4 and GATA-5 methylation profiles in human pancreatic cancers indicate promoter methylation patterns distinct from other an average of 6.8 years between the birth of the cell giving rise to the human tumor types. Cancer Biol. Ther. 6, 1546–1552 (2007). parental clone and the seeding of the index metastasis. Unfortunately, 15. Peiffer, D. A. et al. High-resolution genomic profiling of chromosomal aberrations the great majority of patients are not diagnosed until the last 2 years of using Infinium whole-genome genotyping. Genome Res. 16, 1136–1148 (2006). the entire tumorigenic process. The challenge is to detect these tumours 16. Bignell, G. R. et al. Signatures of mutation and selection in the cancer genome. Nature 463, 893–898 (2010). during time T1, or even after T1 but before seeding of metastases. 17. Sasaki, K. et al. Relationship between labeling indices of KI-67 and brdurd in Advanced imaging methods, as well as blood tests to detect cancer- human malignant tumors. Cancer 62, 989–993 (1988). specific proteins, transcripts or genes12, offer hope for such non-invasive 18. Rew, D. A. & Wilson, G. D. Cell production rates in human tissues and tumours and their significance. Part II: clinical data. Eur. J. Surg. Oncol. 26, 405–417 (2000). early detection. 19. Steel, G. G. The Growth Kinetics of Tumours. (Clarendon Press, Oxford, 1977). 20. Amikura, K., Kobari, M. & Matsuno, S. The time of occurrence of liver metastasis in METHODS SUMMARY carcinoma of the pancreas. Int. J. Pancreatol. 17, 139–146 (1995). Rapid autopsies were performed on seven individuals with Stage IV pancreatic 21. Naumov, G. N. et al. A model of human tumor dormancy: an angiogenic switch from the nonangiogenic phenotype. J. Natl. Cancer Inst. 98, 316–325 (2006). cancer13. Genomic DNA was extracted from cell lines or xenografts established from one metastasis of each patient and used for exomic sequencing as described Supplementary Information is linked to the online version of the paper at previously5. The Illumina Infinium II Whole Genome Genotyping Assay using the www.nature.com/nature. BeadChip platform was also used to analyse each sample at 1,072,820 (1M) SNP Acknowledgements This work was supported by National Institutes of Health grants loci as described previously5. Samples of snap-frozen pancreatic cancer tissue were CA106610 (C.A.I.-D.), CA62924 (C.A.I.-D., M.A.N.), CA43460 (B.V.), CA57345 (K.W.K. microdissected using a PALM MicroLaser System (Carl Zeiss MicroImaging) and and V.E.V.), CA121113 (V.E.V. and K.W.K.), GM078986 (M.A.N.), the Bill and Melinda DNA extracted using QIAamp DNA Micro Kits (Qiagen). Genomic DNA was Gates Foundation Grand Challenges Grant 37874, the Uehara Memorial Foundation (S.Y.), the AACR-Barletta Foundation (C.A.I.-D.), the John Templeton Foundation, Pilot quantified by calculating long interspersed nuclear elements (LINE) by real-time Funding from the Sol Goldman Pancreatic Cancer Research Center, the Michael Rolfe PCR. Whole genome amplification (WGA) was performed using 10 ng total tem- Pancreatic Cancer Foundation, the George Rubis Endowment for Pancreatic Cancer plate DNA and an illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare). Research, the Joseph C. Monastra Foundation for Pancreatic Cancer Research, the Ki-67 immunolabelling (Clone MIB-1, Dako Cytomation) was performed on Alfredo Scatena Memorial Fund, the Virginia and the D.K. Ludwig Fund for Cancer formalin-fixed, paraffin-embedded sections of normal pancreatic ducts and meta- Research, The Joint Program in Mathematical Biology and J. Epstein. We would like to acknowledge T. C. Cornish, C. Henderson, N. Omura and S.-M. Hong for their technical static pancreatic cancer tissues for each patient using the Ventana Discovery stain- assistance in selected aspects of this work. ing system (Ventana Medical Systems), and this information was used to inform computational models of the timing of clonal evolution of each patient’s pancreatic Author Contributions Sample collection and processing was performed by C.A.I.-D., cancer (full details of these models are available in Full Methods). S.Y., B.F. and M.K. Microdissection, DNA extractions and whole genome amplification reactions were performed by S.Y. Sequencing was performed by S.J. Copy number Full Methods and any associated references are available in the online version of analyses were performed by R.L. Computational models and estimates of clonal the paper at www.nature.com/nature. evolution were performed by I.B., T.A. and M.A.N.; C.A.I.-D., S.Y., S.J., R.H.H., J.R.E., M.A.N., I.B., T.A., V.E.V., K.W.K. and B.V. directed the research. C.A.I.-D., B.V., S.Y., I.B. and T.A. wrote the manuscript, which all authors have approved. Received 11 May; accepted 15 September 2010. Author Information Reprints and permissions information is available at 1. Fidler, I. J. Critical determinants of metastasis. Semin. Cancer Biol. 12, 89–96 www.nature.com/reprints. The authors declare no competing financial interests. (2002). Readers are welcome to comment on the online version of this article at 2. Nguyen, D. X., Bos, P. D. & Massague, J. Metastasis: from dissemination to organ- www.nature.com/nature. Correspondence and requests for materials should be specific colonization. Nature Rev. Cancer 9, 274–284 (2009). addressed to C.A.I.-D. ([email protected]).

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METHODS calculated. Normal pancreas was collected from two autopsied patients who died of Patients and tissue samples. Tissue samples from seven patients with pancreatic causes other than pancreatic cancer and five patients who underwent distal pan- ductal adenocarcinoma were collected in association with the Gastrointestinal createctomy for a serous cystadenoma or an islet cell tumour at The Johns Hopkins Cancer Rapid Medical Donation Program (GICRMDP). This programme was Hospital. Paraffin blocks were cut into sections 4-mm thick for Ki-67 immunostain- approved by the Johns Hopkins institutional review board and deemed in accord- ing with all staining processes from deparaffinization to counterstaining with hae- ance with the Health Insurance Portability and Accountability Act. Details of the matoxylin being performed automatically with the Ventana Discovery staining programme have been described in detail previously13. The tissue harvesting pro- system (Ventana Medical Systems). An anti-human Ki-67 mouse monoclonal tocol consists of the following; after opening of the body cavity using standard antibody (Clone MIB-1, Dako Cytomation) was used. At least 12 randomly selected techniques, the whole pancreas including the pancreatic cancer and each grossly high-power fields containing a minimum of 2,000 cells were evaluated for each case, identified metastasis were sampled using a sterile blade and forceps. The whole and the labelling index (LI) was calculated as the percentage of positive cell nuclei. pancreas was sliced into 13 1 3 0.4 cm sections for overnight fixation in 10% buf- Reactive small lymphocytes in each case were regarded as internal positive controls fered-formalin, for freezing in Tissue-Tek OCT compound (Sakura Finetechnical) for Ki-67. Equal or more intense nuclear staining in comparison with the internal in liquid nitrogen and for snap-freezing in liquid nitrogen in 1.7 ml cryovials and positive controls was considered to indicate positivity. storage at 280 uC. Xenograft enriched or low passage cell lines were generated from Modelling tumour evolution. Passenger mutations were defined as those unlikely the post mortem cancer tissues of these seven patients as described previously13,14. to drive tumorigenesis. To be conservative, we considered passenger mutations as Laser capture microdissection (LCM). Frozen tissue sections of autopsy tissues those not included as candidate cancer genes in a recent study based on whole 5 were cut into 7 mm sections using a cryostat and embedded onto UV-treated exome sequencing of 24 pancreatic cancers . As the great majority of mutations PALM membrane slides (Carl Zeiss MicroImaging) and the slides were stored identified in cancers are believed to be passengers, the results of the model are not immediately at 280 uC until subsequent fixation. Tissue sections that underwent highly dependent on the model used to estimate the relatively small number of 16 LCM were defrosted, fixed in 100% methanol for 3 min, and stained with toluidine drivers . blue before microdissection to remove contaminating stromal elements. Sections Because passenger mutations are neutral and do not affect the evolution in any were dissected using a PALM MicroLaser System (Carl Zeiss MicroImaging). way, they are accumulated independently in each cell lineage. Following the Dissected tissues were catapulted into adhesive caps. Generally, .20,000 cells were lineage of the founder cell of the parental clone back in time, we can assume that obtained from 5–10 serial sections by LCM to obtain sufficient quantity and it acquired a new neutral mutation with rate r at each cell division, with r being the quality of genomic DNA for subsequent amplification and sequencing. product of the mutation rate per base pair per cell division and the number of base Genomic DNA extraction and whole genome amplification. Genomic DNA pairs sequenced. The accumulation of neutral mutations in a cell lineage can be from microdissected tissues was extracted using a QIAmp DNA Micro Kit (Qiagen) well-described by a Poisson process with rate r per cell division. We are interested according to the manufacturer’s protocol. Genomic DNA was quantified by cal- in the number of cell divisions in the single lineage between tumour initiation and culating long interspersed nuclear elements (LINE) by real-time PCR. The LINE birth of the founder cell of the parental clone during which N1 passenger mutations primer set 59-AAAGCCGCTCAACTACATGG-39 (forward) and 59-TGCTTTGA accumulate. On the other hand, N1 is also the number of mutations that are found ATGCGTCCCAGAG-39 (reverse) was designed. The real-time PCR conditions in all tumour samples from one patient. Since we sequenced at least one sample were 95 uC for 10 min; 40 cycles of 94 uC for 10 s, 58 uC for 15 s and 70 uC for from the primary tumour and at least three samples from different metastases 30 s. PCR was carried out using Platinum SYBR Green qPCR SuperMix-UDG from each patient, these specific N1 mutations had to be present in the founder (Invitrogen). To minimize sequencing bias from using low-copy starting templates, cells of all three metastases and in cells in the primary tumour. Thus there was a cell only samples for which the measured concentration by LINE assay was$ 3.3 ng in the tumour that had these N1 specific mutations for the first time, and that is, by ml21 (1,000 genome equivalents) were used as a starting template for whole genome definition, the founder cell of the parental clone. Since we can neglect the accu- amplification (WGA). WGA was performed using 10 ng total template DNA and mulation of mutations before the onset of the tumour, these N1 mutations are an illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare), following the accumulated along the single lineage from the tumour initiator cell to the founder manufacturer’s protocol. WGA products were purified using a Microspin G-50 cell of the parental clone. As the number of cell divisions between two subsequent system (GE Healthcare). The purified WGA products were quantified by mutations is distributed according to an exponential distribution with mean 1/r, 21 NanoDrop spectrophotometer (Thermo Fisher Scientific) and diluted to 20 ng ml the required number of cell divisions is the sum of N1 independent exponentially for sequencing analysis. Using these methods and quality controls, there was com- distributed random variables with mean 1/r, and is distributed according to a plete concordance in the mutational signatures obtained of cultured cell lines/ Gamma distribution with shape parameter N1 and scale parameter 1/r. The mean xenografts versus WGA materials prepared from their matched frozen tissues. of this distribution is N1/r and the standard deviation is N1=r (see Sanger sequencing. PCR amplification and sequencing was performed using the Supplementary Table 6). Because the number of base pairs sequenced in the study pffiffiffiffiffiffiffiffiffiffi conditions and primers described previously5. A small number of sequencing reac- is 31.7 3 106, and the mutation rate per base pair per generation is estimated at tions failed (,2% of the total reactions) and these corresponding genes were not 5 3 10210, r 5 31.7 3 106 3 5 3 10210 < 0.016 per generation8. included in progression models or quantitative time estimates of clonal evolution. Using our measurements of Ki-67 labelling index of the seven index lesions Genotyping. The Illumina Infinium II Whole Genome Genotyping Assay using (average 16.3%), we were able to estimate the S-phase fraction of cells in the seven the BeadChip platform was used to analyse tumour samples at 1,072,820 (1M) index lesions (average LI 5 9.5%)17. Assuming a median value for the S-phase 5 SNP loci as previously described . Briefly, all SNP positions were based on the hg18 duration in human tissues and tumours, Ts, of 10 h (ref. 18) and using the formula (NCBI Build 36, March 2006) version of the human genome reference sequence. for the potential cell doubling time Tpot 5 lTs/LI, we get an estimate for Tpot of The genotyping assay begins with hybridization to a 50-nucleotide oligonucleo- 3.5 days. Here l is a correction factor for the nonlinear age distribution of cells tide, followed by a two-colour fluorescent single-base extension. Fluorescence through the cell cycle, which was assumed to be 0.8 (ref. 19). This estimate is intensity image files were processed using Illumina BeadStation software to pro- consistent with the average cell doubling time in pancreatic cancer from ref. 20 of vide normalized intensity values and allelic frequency for each SNP position. For 2.3 days. We use this latter estimate in our analysis, as we believe it is more accurate each SNP, the normalized experimental intensity value (R) was compared to the for pancreatic cancer. intensity values for that SNP from a training set of normal samples and repre- Our model works very well for estimating the number of cell divisions between sented as a ratio (called the ‘log R ratio’) of log2 (Rexperimental/Rtraining set). For each discrete events in tumour evolution. In order to go from number of cell divisions to SNP, the normalized allele intensity ratio (theta) was used to estimate a quant- actual time we need to have an estimate for the average rate of cell division. The itative allelic frequency value (called the ‘B allele frequency’) for that SNP15. Using accuracy of our predictions regarding actual time therefore depends on the accu- Illumina BeadStudio software, log R ratio and B allele frequency values were racy of that estimate. If we let Tgen denote the average time between subsequent cell plotted along chromosomal coordinates and examined visually. Regions of loss divisions in a cell lineage, we arrive at the expression for time T1: of heterozygosity (LOH) were identified as genomic regions .2 megabases (Mb) Tgen with consecutive homozygous genotype calls (B allele frequency near 0 or 1). T1~ (N1+pN1): Smaller (,2 Mb) regions of LOH were identified by requiring co-occurrence of r decreased log R ratio scores in regions of consecutive homozygous genotype calls We therefore estimate the number of cell divisions,ffiffiffiffiffiffi and hence the time T1 (B allele frequency near 0 or 1). Visual analysis of these data plotted along chromo- between tumour initiation and birth of the founder cell of the parental clone, to somal coordinates was followed by manual analysis of the data for selected genes of be proportional to the number of passenger mutations, N1, that the tumour interest. acquired during that time. In our calculations, we use the estimate for cell doubling 20 Estimations of proliferation rates. To estimate the cell division rate, the Ki-67 time in pancreatic cancer from the literature as the value of Tgen. labelling index (LI) in the proband lesion for each case was calculated. The Ki-67 LI T2 is determined analogously, with N2 defined as the number of passenger on the pancreatic ducts in the histologically normal pancreas parenchyma was also mutations present in the index lesion but not in the parental clone. T3 is

©2010 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH determined from literature-based estimates of the tumour and cell doubling times, We estimated the tumour doubling time was equal to the cell doubling time 20 and the size of the index lesions at autopsy . (Tgen) until the tumour size reached 100 mm in diameter at which time angio- The median doubling time of pancreatic cancer metastases was reported as genesis is required21. Thereafter, we used the median doubling time described 56 days20. To estimate the age of the index metastasis, we used a two stage model. above.

©2010 Macmillan Publishers Limited. All rights reserved SUPPLEMENTARY INFORMATION doi:10.1038/nature09515



   


 







  
 

 
  



 

 
  

  
 


 

   





 




  

  


  

  


 





 

  
  
  

   
  
 




  


 
 

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WWW.NATURE.COM/ NATURE | 3 3 NEWS & VIEWS RESEARCH its current level around 700 million years ago, permanently revved up — that is, until rooted- begin to develop informed hypotheses about coinciding with the cessation of snowball ness came into play several hundred million how variations in the phosphorus cycle are Earth conditions. This peak reflects a large years later. A test of this hypothesized mecha- driven, and what impact they have on the increase in the marine phosphorus inventory nism of enhanced phosphorus stripping from global carbon cycle, oxygen levels and the that was sustained over tens of millions of landscapes would be to identify deltaic or other evolution of marine ecosystems. O years. Given that ‘modern’ marine phospho- sedimentary-basin environments from the rus has a residence time of tens of thousands late Proterozoic, and to use proxy estimates of Gabriel M. Filippelli is in the Department of years, it is hard to imagine ocean conditions phosphorus loss (for example, the phosphorus/ of Earth Sciences, Indiana University– in which sustained high levels of phosphorus aluminium ratio) to determine whether values Purdue University Indianapolis (IUPUI), were not driven by a step-change in the global for this time interval are lower than expected Indianapolis, Indiana 46202, USA. phosphorus mass balance. given the source material. Such analyses e-mail: [email protected] The thinking about the consequences of would provide independent evidence of high 1. Catling, D. C., Glein, C. R., Zahnle, K. J. & McKay, C. P. such high phosphorus levels then runs as fol- phosphorus weathering rates. Astrobiology 5, 415–438 (2005). 4 lows. The result of the phosphorus-driven Meanwhile, with this paper and use of the 2. Hoffman, P. F., Kaufmann, A. J., Halverson, G. P. & marine productivity was sustained algal phosphorus/iron proxy, there is now another Schrag, D. P. Science 281, 1342–1346 (1998). 3. Hoffman, P. F. & Schrag, D. P. Terra Nova 14, blooms in the ocean, much like those found way to look at nutrient variations in the ancient 129–155 (2002). today in ponds and streams near areas of heavy oceans. No proxy is perfect, however. In this 4. Planavsky, N. J. et al. Nature 467, 1088–1090 fertilizer application. The death and settling instance, the weaknesses are both obvious (2010). of these blooms caused long-term, enhanced (the limited spatial and temporal range of the 5. Tyrell, T. Nature 400, 525–531 (1999). 6. Mort, H. P. et al. Geology 35, 483–486 (2007). organic-carbon burial, which (via the mass- proper rock types for analysis) and less obvious 7. Paytan, A. & McLaughlin, K. Chem. Rev. 107, balance relationship between carbon and (variations in original iron-oxide composition 563–576 (2007). oxygen9) resulted in the addition of oxygen to that are now masked by mineral matura- 8. Diaz, J. et al. Science 320, 652–655 (2008). the ocean–atmosphere system (Fig. 1). This tion). Nevertheless, thanks to Planavsky and 9. Berner, R. A. Nature 426, 323–326 (2003). 4 10. Föllmi, K. B., Hosein, R., Arn, K. & Steinmann, P. increase in atmospheric oxygen controlled the colleagues , we have a picture of the marine Geochim. Cosmochim. Acta 73, 2252–2282 (2009). evolutionary patterns of oxygen-dependent phosphorus cycle through deep time. We can 11. Filippelli, G. M. et al. Quat. Res. 66, 158–166 (2006). metazoans. Such a scenario provides a plau- sible link between the roles of snowball Earth glaciations and late Proterozoic oxygena- CANCER tion in leading to the explosion in metazoan diversity. The implications of the new results4 for understanding glacially induced phospho- Genomic evolution rus weathering on landscapes are also inter- esting. We are beginning to appreciate how glacial dynamics affects phosphorus weather- of metastasis ing on land and transport to the oceans10. In the modern ‘rooted’ world, in which soil devel- Prognosis for patients with pancreatic cancer is bleak, often owing to late diagnosis. opment is generally mediated by plants, most The estimate that at least 15 years pass from tumour initiation to malignancy offers of the weathered phosphorus is mobilized hope for early detection and prevention. S L .1109  .1114 and transported from landscapes in a narrow time window after a glacier retreats. Conti- nental records11 indicate that this large flux E. GEORG LUEBECK rearrangements. They find that specific chro- of phosphorus occurs in about 10,000 years mosomal rearrangements known as fold-back in most landscapes (probably faster in low- adiocarbon dating and comparative inversions occur in almost all of a patient’s relief/high-rainfall landscapes and slower analyses of skeletal anatomy have metastatic lesions. What’s more, unlike other in high-relief/low-rainfall landscapes). In a informed the theory of human evolu- chromosomal rearrangements, which seem to modern landscape with its considerable plant Rtion; similarly, DNA sequencing of tumour occur either in the primary, parental tumour coverage, maintenance of a sustained increase cells coupled with dissection of the molecular or in the metastatic lesion, Campbell et al. in phosphorus delivery to the oceans would anatomy of chromosomal aberrations is begin- detect fold-back inversions in both primary require periodic removal of weathered and ning to yield deeper insight into the evolution and metastatic tumours. The authors therefore phosphorus-depleted soils, and exposure of of cancer. In this issue, two papers1,2 present argue that fold-back inversions occur early in fresh material for further soil development. findings from sequencing the protein-coding tumorigenesis and are probably a crucial driver In late Proterozoic time, however, the regions (exons) of more than 20,000 genes of pancreatic-cancer progression. absence of land plants meant that there would from the genomes of patients with metastatic The precise origin of fold-back inversions have been little soil development to stabilize (stage IV) pancreatic cancer. The findings is unknown. It could be that DNA-replica- landscapes, or to convert mineral-based phos- are unprecedented, providing the first high- tion-related erosion of the telomeres (the phorus forms on pristine mineral surfaces to resolution image (at the level of single chromosome ends) — potentially because the organically and oxide-bound phosphorus base pairs) of the non-germline mutational of suppressed or dysfunctional activity of found in modern soils. Presumably, phos- spectrum of pancreatic tumours and their the enzyme telomerase — triggers recurrent phorus stripping from rocks was much more metastatic descendants. breakage–fusion–bridge (B/F/B) cycles3,4, extensive, as the landscapes themselves were It has long been recognized that genomic which, in turn, cause progressive gains and much less stabilized because of the lack of instability is a hallmark of cancer. However, its losses of genetic material and so genomic flora. Thus, in this case, the present is not a significance in cancer progression has been the instability. Intriguingly, telomerase activity key to the distant past. Without the systems subject of debate for just as long. To shed light seems to be restored in the invasive tumours, to stabilize phosphorus, the phosphorus on this, Campbell et al.1 (page 1109) performed which might have a stabilizing effect on the cycle in the late Proterozoic would have been a DNA-sequence-based study of chromosomal abundance of B/F/B-induced rearrangements,

28 O C TOB ER 2010 | VOL 467 | NATURE | 1053 © 2010 Macmillan Publishers Limited. All rights reserved NEWS & VIEWS RESEARCH

that tissue. Thus, Yachida and colleagues’ esti- mate must be considered a lower bound for Non-metastatic primary tumour Metastatic subclones Metastases the mean sojourn time of pancreatic lesions, such as pancreatic intraepithelial neoplasia, that have the potential to cause invasive and metastatic cancer. From a clinical perspective, what matters is the prospective disease risk, which may involve multiple lesions individu- ally evolving towards cancer. Thus, the time estimates of Yachida et al. are conservative and so clinically relevant. 1,2 > 10 years ~5–6 years ~3 years These two studies are a bellwether, and are among the first to explore the biological Tumour Parental Metastatic Patient’s and clinical implications of sequence data for initiation clone seeds death individual tumours. As the sequencing tech- nology moves forward — and it does so at a blinding speed — more exciting details of the Figure 1 | The pancreatic-cancer timeline. Mathematical analyses of tumour-DNA sequence data 1,2 evolutionary pro cesses involved in tumour from two collaborative studies suggest that it probably takes more than 10 years from the initiation of a progression are likely to be unearthed. It is pancreatic tumour to the birth of the parental clone that results in pancreatic cancer. However, this clone to be hoped that such information will not does not have metastatic potential, and the subclones with the ability to spread to other tissues develop over an additional 5–6 years. The metastases, which are soon followed by the patient’s death, occur over only deepen our understanding of the cancer roughly the next 3 years. pro cess, but also lead to new approaches to early cancer detection, better prognosis and, but not on other rearrangements. earlier, population-based analysis5 estimated ultimately, prevention. O Campbell and colleagues’ results affirm the that the mean sojourn time from the tumour- presence of genomic instability in the devel- initiating mutation to clinical diagnosis E. Georg Luebeck is at the Fred Hutchinson opment of pancreatic cancer. But because of may be as much as five to six decades. Cancer Research Center, Program in extensive differences in the number, type and On the surface, the population-based esti- Computational Biology, Seattle, Washington position of the rearrangements among patients mate seems much longer than Yachida et al. 98109-1024, USA. — and even between the metastatic deposits conclude. It should be kept in mind, however, e-mail: [email protected] in the same organ of a single patient — the that the present sequence-based time esti- 2 1. Campbell, P. J. et al. Nature 467, 1109–1113 (2010). functional consequences of this instability mates are not general: they do not refer to the 2. Yachida, S. et al. Nature 467, 1114–1117 (2010). remain unclear. Studies using next-generation average of all pancreatic lesions with cancerous 3. McClintock, B. Genetics 26, 234–282 (1941). 4. Lo, A. W. I. et al. Neoplasia 4, 531–538 (2002). sequencing technologies on a larger number of and metastatic potential in the tissue, but rather 5. Meza, R., Jeon, J., Moolgavkar, S. H. & Luebeck, patients are likely to fill in the missing pieces refer to the one lesion in the tissue which, by E. G. Proc. Natl Acad. Sci. USA 105, 16284–16289 and pinpoint the driving forces in tumour pro- chance, leads to the first primary tumour in (2008). gression and metastatic dissemination across different types of cancer. In a separate study, Yachida et al.2 STEM CELLS (page 1114) address the clinically relevant issue of the timescales associated with tumour pro- gression. These authors also carry out genomic sequencing of pancreatic-cancer metastases The intestinal-crypt and examine their phylogenetic relationship with their respective, previously sequenced, primary tumours in seven patients. They casino thus derive estimates of three timescales: the time from tumour initiation to the birth of the Stem cells can renew themselves indefinitely — a feature that is often attributed founder cell of the parental (non-metastatic) to asymmetrical cell division. Fresh experimental and mathematical models of clone; the sojourn time between the paren- the intestine provide evidence that begs to differ. tal clone arising and its acquisition of meta- static potential; and the time from metastatic dissemination to the patient’s death (Fig. 1). MICHAEL P. VERZI & RAMESH A. SHIVDASANI in principle there is no reason why individual Remarkably, the authors estimate that the stem cells should not divide symmetrically, to time from tumour initiation to metastatic ertain tissues, such as the skin, blood generate either two identical stem cells or two dissemination is at least a decade — a con- and intestinal lining, replenish millions daughters that exit the pool to differentiate. clusion that suggests that there is a window of lost cells every day. The burden of Indeed, two reports published in Science3 and of opportunity for medical intervention Crenewal falls on small populations of stem cells, Cell4 demonstrate that, in the normal course before the cancer spreads to distant organs. which can make exact copies of themselves, as of tissue renewal, intestinal stem cells divide This finding is not inconsistent with that well as generate all the resident cell types that symmetrically. inferred from quantitative analyses of the age- differentiate and eventually die. This rare dual In the small intestine, stem cells lie at visually specific incidence of pancreatic cancer in the ability, a defining property of all stem cells, is identifiable positions within pocket-like general population5. On the basis of a general exemplified by the asymmetrical division of crypts, and their progeny migrate in predict- mathematical description that recognizes the germ cells1 and neuronal precursors2 in the able streams (Fig. 1a, overleaf). In mice, two random nature of both mutation accumulation fruitfly. Nonetheless, as long as the total stem- cell populations manifest the capacity for both and clonal expansion in pancreatic cancer, the cell pool in a tissue remains roughly constant, prolonged self-renewal and multi-lineage

28 O C TOB ER 2010 | VOL 467 | NATURE | 1055 © 2010 Macmillan Publishers Limited. All rights reserved