LETTER doi:10.1038/nature10738 Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing Li Ding.2+,Timothy J.Ley.3.4*,David E.Larson',Christopher A.Miller,Daniel C.Koboldt',John S.Welch3,Julie K.Ritchey3, Margaret A.Young Tamara Lamprecht,Michael D.McLellan',Joshua F.McMichael',John W.Wallis2,Charles Lu',Dong Shen' Christopher C.Harris David J.Dooling 2,Robert S.Fulton,Lucinda L.Fulton2,Ken Chen2,Heather Schmidt', Joelle Kalicki-Veizer,Vincent J.Magrini2,Lisa Cook',Sean D.McGrath',TammiL.Vickery',MichaelC.Wendl2,Sharon Heath2 Mark A.WatsonDanielC.Link4,Michael H.Tomasson3.4,William D.Shannon,Jacqueline E.Payton,Shashikant Kulkarni2.4.5 Peter Westervelt Matthew J.WalterTimothy A.GraubertElaineR.MardisRichard K.Wilson&JohnF.DiPersio Most patients with acute myeloid leukaemia(AML)die from pro- 1 to 3 were validated (see ref.7 for tier designations;Supplementary gressive disease after relapse,which is associated with clonal evolu- Fig.1a and Supplementary Tables 4a and 5).Of these,78 mutations tion at the cytogenetic level2.To determine the mutational were relapse-specific(63 point mutations,1 dinucleotide mutation,13 spectrum associated with relapse,we sequenced the primary tumour indels and 1 translocation;relapse-specific criteria described in Sup- and relapse genomes from eight AML patients,and validated plementary Information and shown in Supplementary Fig 1b),5 point hundreds of somatic mutations using deep sequencing;this allowed mutations were primary-tumour-specific,and 330(317 point mutations us to define clonality and clonal evolution patterns precisely at and 13 indels)were shared between the primary tumour and relapse relapse.In addition to discovering novel,recurrently mutated genes samples (Fig.1a,b and Supplementary Fig.2).The skin sample was (for example,WAC SMC3,DIS3,DDX4I and DAXX)in AML,we contaminated with leukaemic cells for this case (peripheral white blood also found two major clonal evolution patterns during AML relapse: cell count was 105,000 cells mm3 when the skin sample was banked), (1)the founding clone in the primary tumour gained mutations and with an estimated tumour content in the skin sample of 29% evolved into the relapse clone,or(2)a subclone of the founding (Supplementary Information).In addition to the ten somatic non- clone survived initial therapy,gained additional mutations and synonymous mutations originally reported for the primary tumour expanded at relapse.In all cases,chemotherapy failed to eradicate sample,we identified one deletion that was not detected in the original the founding clone.The comparison of relapse-specific versus analysis(DNMT3A L723fs(ref.8))and three mis-sense mutations previ- primary tumour mutations in all eight cases revealed an increase ously misclassified as germline events(SMC3 G662C,PDXDCI E421K in transversions,probably due to DNA damage caused by cytotoxic and TTNE14263K)(Fig.1b,Table 1 and Supplementary Table 4b). chemotherapy.These data demonstrate that AML relapse is asso- A total of 169 tier 1 coding mutations(approximately 21 per case) ciated with the addition of new mutations and clonal evolution, were identified in the eight patients (Table 1 and Supplementary which is shaped,in part,by the chemotherapy that the patients Tables 4b and 6),of which 19 were relapse-specific.In addition to receive to establish and maintain remissions. mutations in known AML genes such as DNMT3A(ref.8),FLT3 To investigate the genetic changes associated with AML relapse,and (ref.9),NPMI(ref.10),IDHI(ref.7),IDH2(ref.11),WTI(ref.12) to determine whether clonal evolution contributes to relapse,we per- RUNXI(refs 13,14),PTPRT(ref.3),PHF6(ref.15)and ETV6(ref.16) formed whole-genome sequencing of primary tumour-relapse pairs in these eight patients,we also discovered novel,recurring mutations and matched skin samples fromeight patients,including unique patient in WAC,SMC3,DIS3,DDX41 and DAXX using 200 AML cases whose identifier(UPN)933124,whose primary tumour mutations were previ- exomes were sequenced as part of the Cancer Genome Atlas AML ously reported.Informed consent explicit for whole-genome sequen- project(Table 1,Supplementary Table 4b and Supplementary Fig.3; cing was obtained for all patients on a protocol approved by the T.J.L.,R.K.W.and The Cancer Genome Atlas working group on AML, Washington University Medical School Institutional Review Board. unpublished data).Details regarding the novel,recurrently mutated We obtained >25X haploid coverage and >97%diploid coverage for genes are provided in Table 1,Supplementary Tables 4b and 7 and each sample(Supplementary Table 1 and Supplementary Information). Supplementary Figs 3 and 4.Structural and functional analyses of These patients were from five different French-American-British structural variants are presented in the Supplementary Information haematological subtypes,with elapsed times of 235-961 days between (Supplementary Figs 5-10 and Supplementary Tables 2,8 and 9). initial diagnosis and relapse(Supplementary Table 2a,b). The generation ofhigh-depth sequencing data allowed us to quantify Candidate somatic events in the primary tumour and relapse genomes accurately mutant allele frequencies in all cases,permitting estimation were identifiedss and selected for hybridization capture-based validation of the size of tumour clonal populations in each AML sample.On the using methods described in Supplementary Information.Deep sequen- basis of mutation clustering results,we inferred the identity of four cing of the captured target DNAs from skin (the matched normal tissue), clones having distinct sets of mutations (clusters)in the primary primary tumour and relapse tumour specimense (Supplementary tumour of AML1/UPN 933124(Supplementary Information).The Table 3)yielded a median of 590-fold coverage per site.The average median mutant allele frequencies in the primary tumour for clusters number of mutations and structural variants was 539(range 118- 1to 4 were 46.86%,24.89%,16.00%and 2.39%,respectively (Fig.1band 1,292)per case (Fig.1a). Supplementary Table 5c).Clone 1 is the 'founding'clone(that is,the The general approach for relapse analysis is exemplified by the first other subclones are derived from it),containing the cluster I mutations; sequenced case(UPN 933124).A total of 413 somatic events from tiers assuming that nearly all of these mutations are heterozygous,they must The Genome Institute,Washington University,St Louis,Missouri63108,USA2Department of Genetics,Washington University,StLouis,Missouri63110,USADepartmentof Internal Medicine,Division of Oncology,Washington University,St Louis,Missouri63110.USASiteman Cancer Center,Washington University,St Louis Missouri63110.USASDepartment of Pathology and Immunology.Washington University,St Louis,Missouri 63110,USA Division of Biostatistics,Washington University,St Louis,Missouri 63110,USA. .These authors contributed equally to this work 506 NATURE I VOL JANUARY 2012 2012 Macmillan Publishers Limited.All rights reserved
LETTER doi:10.1038/nature10738 Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing Li Ding1,2*, Timothy J. Ley1,3,4*, David E. Larson1 , Christopher A. Miller1 , Daniel C. Koboldt1 , John S. Welch3 , Julie K. Ritchey3 , Margaret A. Young3 , Tamara Lamprecht3 , Michael D. McLellan1 , Joshua F. McMichael1 , John W. Wallis1,2, Charles Lu1 , Dong Shen1 , Christopher C. Harris1 , David J. Dooling1,2, Robert S. Fulton1,2, Lucinda L. Fulton1,2, Ken Chen1,2, Heather Schmidt1 , Joelle Kalicki-Veizer1 , Vincent J. Magrini1,2, Lisa Cook1 , Sean D. McGrath1 , Tammi L. Vickery1 , Michael C.Wendl1,2, Sharon Heath3 , Mark A.Watson5 , Daniel C. Link3,4, Michael H. Tomasson3,4,William D. Shannon6 , Jacqueline E. Payton5 , Shashikant Kulkarni2,4,5, Peter Westervelt3,4, Matthew J.Walter3,4, Timothy A. Graubert3,4, Elaine R. Mardis1,2,4, Richard K. Wilson1,2,4 & John F. DiPersio3,4 Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level1,2. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns duringAML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions. To investigate the genetic changes associated with AML relapse, and to determine whether clonal evolution contributes to relapse, we performed whole-genome sequencing of primary tumour–relapse pairs and matched skin samples from eight patients, including unique patient identifier (UPN) 933124, whose primary tumour mutations were previously reported3 . Informed consent explicit for whole-genome sequencing was obtained for all patients on a protocol approved by the Washington University Medical School Institutional Review Board. We obtained .253 haploid coverage and .97% diploid coverage for each sample (Supplementary Table 1 and Supplementary Information). These patients were from five different French–American–British haematological subtypes, with elapsed times of 235–961 days between initial diagnosis and relapse (Supplementary Table 2a, b). Candidate somatic events in the primary tumour and relapse genomes were identified4,5 and selected for hybridization capture-based validation using methods described in Supplementary Information. Deep sequencing of the captured target DNAsfrom skin (the matched normal tissue), primary tumour and relapse tumour specimens6 (Supplementary Table 3) yielded a median of 590-fold coverage per site. The average number of mutations and structural variants was 539 (range 118– 1,292) per case (Fig. 1a). The general approach for relapse analysis is exemplified by the first sequenced case (UPN 933124). A total of 413 somatic events from tiers 1 to 3 were validated (see ref. 7 for tier designations; Supplementary Fig. 1a and Supplementary Tables 4a and 5). Of these, 78 mutations were relapse-specific (63 point mutations, 1 dinucleotide mutation, 13 indels and 1 translocation; relapse-specific criteria described in Supplementary Information and shown in Supplementary Fig. 1b), 5 point mutations were primary-tumour-specific, and 330 (317 pointmutations and 13 indels) were shared between the primary tumour and relapse samples (Fig. 1a, b and Supplementary Fig. 2). The skin sample was contaminated with leukaemic cells for this case (peripheral white blood cell count was 105,000 cells mm23 when the skin sample was banked), with an estimated tumour content in the skin sample of 29% (Supplementary Information). In addition to the ten somatic nonsynonymous mutations originally reported for the primary tumour sample3 , we identified one deletion that was not detected in the original analysis (DNMT3AL723fs (ref. 8)) and three mis-sense mutations previously misclassified as germline events (SMC3 G662C, PDXDC1 E421K and TTN E14263K) (Fig. 1b, Table 1 and Supplementary Table 4b). A total of 169 tier 1 coding mutations (approximately 21 per case) were identified in the eight patients (Table 1 and Supplementary Tables 4b and 6), of which 19 were relapse-specific. In addition to mutations in known AML genes such as DNMT3A (ref. 8), FLT3 (ref. 9), NPM1 (ref. 10), IDH1 (ref. 7), IDH2 (ref. 11), WT1 (ref. 12), RUNX1 (refs 13, 14), PTPRT (ref. 3), PHF6 (ref. 15) and ETV6 (ref. 16) in these eight patients, we also discovered novel, recurring mutations in WAC, SMC3, DIS3, DDX41 and DAXX using 200 AML cases whose exomes were sequenced as part of the Cancer Genome Atlas AML project (Table 1, Supplementary Table 4b and Supplementary Fig. 3; T.J.L., R.K.W. and The Cancer Genome Atlas working group on AML, unpublished data). Details regarding the novel, recurrently mutated genes are provided in Table 1, Supplementary Tables 4b and 7 and Supplementary Figs 3 and 4. Structural and functional analyses of structural variants are presented in the Supplementary Information (Supplementary Figs 5–10 and Supplementary Tables 2, 8 and 9). The generation of high-depth sequencing data allowed us to quantify accurately mutant allele frequencies in all cases, permitting estimation of the size of tumour clonal populations in each AML sample. On the basis of mutation clustering results, we inferred the identity of four clones having distinct sets of mutations (clusters) in the primary tumour of AML1/UPN 933124 (Supplementary Information). The median mutant allele frequencies in the primary tumour for clusters 1 to 4 were 46.86%, 24.89%, 16.00% and 2.39%, respectively (Fig. 1b and Supplementary Table 5c). Clone 1 is the ‘founding’ clone (that is, the other subclones are derived from it), containing the cluster 1 mutations; assuming that nearly all of these mutations are heterozygous, they must 1 The Genome Institute, Washington University, St Louis, Missouri 63108, USA. 2 Department of Genetics, Washington University, St Louis, Missouri 63110, USA. 3 Department of Internal Medicine, Division of Oncology, Washington University, St Louis, Missouri 63110, USA. 4 Siteman Cancer Center, Washington University, St Louis, Missouri 63110, USA. 5 Department of Pathology and Immunology, Washington University, St Louis, Missouri 63110, USA. 6 Division of Biostatistics, Washington University, St Louis, Missouri 63110, USA. *These authors contributed equally to this work. 506 | NATURE | VOL 481 | 26 JANUARY 2012 ©2012 Macmillan Publishers Limited. All rights reserved
LETTER RESEARCH 1,400 ■Relapse specific 100 1.292 Cluster ID ■Primary tumour specific AML1/UPN933124 1 1.200 ■Shared ·2 1.000 oOutlier B00 00 6 658 AML1AUPN933124 600 498 412 0 400 07 200 150 118 10 20 40 60 0 100 Primary tumour variant allele frequency(%) 845%purity 452198 573988 400220 933124758188804188 42698089586 93.7%purity Cases AML43/UPN869586 AML28/UPN426980 AI31几PNA521Q8 AML15/UPN758168 60 Cluster Cluster Cluste Cluste 号 ●1 号 ●1 12 40 2 ●2 40 2 3 3 ●3 3 0 Primary tumour Relapse Primary tumour Relaose Primary tumour Relapse Primary tumour Relaose 90.8%purity 40.0%purity 90.8%purity 82.6%purity 90.8%purity 36.0%purity 90.8%purity 89.6%purity AML40/UPN804168 AML35/PN573988 A1L27LUP400220 60 Cluster 0 Cluster Cluster ●1 1 ●2 ●2 =2 20 0 Primary tumour Relaose Primary tumour Relapse Pnmary tumour Relapse 90.4%purity 78.6%purity 83.4%purity 28.6%purity 89.2%purity 73.2%purity Figure 1 Somatic mutations quantified by deep sequencing of capture in the primary tumour,and one was found at relapse.Five low-level mutations validation targets in eight acute myeloid leukaemia primary tumour and in both the primary tumour and relapse(including four residing in known copy relapse pairs.a,Summary of tier 1-3 mutations detected in eight cases(not number variable regions)were excluded from the clustering analysis.Non- including translocations).All mutations shown were validated using capture synonymous mutations from genes that are recurrently mutated in AML are followed by deep sequencing.Shared mutations are in grey,primary tumour- shown.The change of mutant allele frequencies for mutations from the five specific mutations in blue and relapse-specific mutations in red.The total clusters is shown(right)between the primary tumour and relapse.The orange number of tier 1-3 mutations for each case is shown above the light-grey and red lines are superimposed.c,The mutation clusters detected in the rectangle.b,Mutant allele frequency distribution of validated mutations from primary tumour and relapse samples from seven additional AML patients.The tier 1-3 in the primary tumour and relapse of case UPN 933124(left).Mutant relationship between clusters in the primary tumour and relapse samples are allele frequencies for five primary-tumour-specific mutations were obtained indicated by lines linking them. froma454 deep read-count experiment.Four mutation clusters were identified Table 1|Coding mutations identified in eight primary tumour-relapse pairs UPN Total tier I mutations Recurrently mutated genes Relapse-specific non-synonymous (primary/relapse) in primary tumour somatic mutations 452198 ©© DNMT3A.NPM1.FLT3.IDHI None 573988 6/8 NPM1.IDH2 STOX2 804168 22/26 FLT3,WT1,PHF6.FAM5C,TTC39A SLC25A12.RIPK4,ABCD2 933124 14/17 DNMT3A,NPM1.FLT3.TTN.SMC3.PTPRT ETV6*.MY018B*.WAC*+.STK4 400220 12/13 FLT3,RUNX1,WT1,PLEKHHI None 426980 32/35 IDH2.MYO1F.DDX4 GBP4.DCLKI.IDH2*.DCLK1*.ZNF260 758168 15/19 DNAH9,DIS3,CNTN5,PML-RARAt ENSG00000180144,DAGLA* 869586 51/50 RUNX1.WT1,TTN.PHF6,NF1.SUZ12,NCOA7,EED,DAXX,ACSS3,WAC,NUMAI None Tier 1 mutation counts exclude RNA genes Recu rent mutati ring in mple Translocations were not 1 mutation counts. 26 JANUARY 2012 VOL 481 NATURE I 507 2012 Macmillan Publishers Limited.All rights reserved
c 0 20 40 60 Variant allele frequency (%) 0 20 40 60 Variant allele frequency (%) 0 20 40 60 Variant allele frequency (%) 0 20 40 60 Variant allele frequency (%) 0 20 40 60 Variant allele frequency (%) 0 20 40 60 Variant allele frequency (%) 0 20 40 60 Variant allele frequency (%) 1 2 Primary tumour 89.2% purity Relapse 73.2% purity AML27/UPN400220 1 2 3 Primary tumour 90.8% purity Relapse 89.6% purity AML15/UPN758168 1 2 3 4 Primary tumour 90.8% purity Relapse 36.0% purity AML31/UPN452198 1 2 3 4 5 Primary tumour 90.8% purity Relapse 82.6% purity AML28/UPN426980 1 Cluster Cluster Cluster Cluster Cluster Cluster Cluster 2 3 4 Primary tumour 90.8% purity Relapse 40.0% purity 1 2 Primary tumour 90.4% purity Relapse 78.6% purity AML40/UPN804168 1 2 Primary tumour 83.4% purity Relapse 28.6% purity AML35/UPN573988 AML43/UPN869586 a b Variant allele frequency (%) 0 20 40 60 80 100 Primary tumour 93.7% purity Relapse 84.5% purity AML1/UPN933124 Primary tumour variant allele frequency (%) Relapse variant allele frequency (%) 0 20 40 60 80 100 0 20 40 60 80 100 AML AML1/UPN933124 1 / UPN933124 DNMT3A (L723fs) ETV6 (R105P) FLT3 (594in_frame_ins) MYO18B (A2317T) NPM1 (W287fs) PTPRT (P1232L) SMC3 (G662C) TTN (E14263K) Cluster ID 1 2 3 4 5 Outlier 118 150 307 412 496 658 882 1,292 0 200 400 600 800 1,000 1,200 1,400 Tier 1 Tier 2 Tier 3 Tier 1 Tier 2 Tier 3 Tier 1 Tier 2 Tier 3 Tier 1 Tier 2 Tier 3 Tier 1 Tier 2 Tier 3 Tier 1 Tier 2 Tier 3 Tier 1 Tier 2 Tier 3 Tier 1 Tier 2 Tier 3 452198 573988 400220 933124 758168 804168 426980 869586 Number of mutations Cases Relapse specific Primary tumour specific Shared Figure 1 | Somatic mutations quantified by deep sequencing of capture validation targets in eight acute myeloid leukaemia primary tumour and relapse pairs. a, Summary of tier 1–3 mutations detected in eight cases (not including translocations). All mutations shown were validated using capture followed by deep sequencing. Shared mutations are in grey, primary tumourspecific mutations in blue and relapse-specific mutations in red. The total number of tier 1–3 mutations for each case is shown above the light-grey rectangle. b, Mutant allele frequency distribution of validated mutations from tier 1–3 in the primary tumour and relapse of case UPN 933124 (left). Mutant allele frequencies for five primary-tumour-specific mutations were obtained from a 454 deep read-count experiment. Four mutation clusters were identified in the primary tumour, and one was found at relapse. Five low-level mutations in both the primary tumour and relapse (including four residing in known copy number variable regions) were excluded from the clustering analysis. Nonsynonymous mutations from genes that are recurrently mutated in AML are shown. The change of mutant allele frequencies for mutations from the five clusters is shown (right) between the primary tumour and relapse. The orange and red lines are superimposed. c, The mutation clusters detected in the primary tumour and relapse samples from seven additional AML patients. The relationship between clusters in the primary tumour and relapse samples are indicated by lines linking them. Table 1 | Coding mutations identified in eight primary tumour–relapse pairs UPN Total tier 1 mutations (primary/relapse) Recurrently mutated genes in primary tumour Relapse-specific non-synonymous somatic mutations 452198 9/9 DNMT3A, NPM1, FLT3, IDH1 None 573988 6/8 NPM1, IDH2 STOX2 804168 22/26 FLT3, WT1, PHF6, FAM5C, TTC39A SLC25A12, RIPK4, ABCD2 933124 14/17 DNMT3A, NPM1, FLT3, TTN, SMC3, PTPRT ETV6*, MYO18B*, WAC*{, STK4 400220 12/13 FLT3, RUNX1, WT1, PLEKHH1 None 426980 32/35 IDH2, MYO1F, DDX4 GBP4, DCLK1, IDH2*, DCLK1*, ZNF260 758168 15/19 DNAH9, DIS3, CNTN5, PML-RARA{ ENSG00000180144, DAGLA* 869586 51/50 RUNX1, WT1, TTN, PHF6, NF1, SUZ12, NCOA7, EED, DAXX, ACSS3, WAC, NUMA1 None Tier 1 mutation counts exclude RNA genes. *Recurrent mutations occurring in relapse sample. { Translocations were not included in tier 1 mutation counts. LETTER RESEARCH 26 JANUARY 2012 | VOL 481 | NATURE | 507 ©2012 Macmillan Publishers Limited. All rights reserved
RESEARCH LETTER be present in virtually all the tumour cells at presentation and at relapse, containing all of the cluster 5 mutations was detected in the relapse as the variant frequency of these mutations is~40-50%.Clone 2(with sample;clone 5 evolved from clone 4,but gained 78 new somatic cluster 2 mutations)and clone 3(with cluster 3 mutations)must be alterations after sampling at day 170.As all mutations in clone5 appear derived from clone 1,because virtually all the cells in the sample contain to be present in all relapse tumour cells,we suspect that one or more of the cluster 1 mutations(Fig.2a).It is likely that a single cell from clone 3 the mutations in this clone provided a strong selective advantage that gained a set of mutations(cluster 4)to form clone 4:these survived contributed to relapse.The ETV6 mutation,the MYO18B mutation, chemotherapy and evolved to become the dominant clone at relapse. and/or the WNKI-WAC fusion are the most likely candidates,as We do not know whether any of the cluster 4 mutations conferred ETV6,MYO18B and WAC are recurrently mutated in AML. chemotherapy resistance;although none had translational consequences, We evaluated the mutation clusters in the seven additional primary we cannot rule out a relevant regulatory mutation in this cluster. tumour-relapse pairs by assessing peaks of allele frequency using Assuming that all the mutations detected are heterozygous in the kernel density estimation (Supplementary Fig.11 and Supplemen- primary tumour sample (with a malignant cellular content at 93.72% tary Information).We thus inferred the numbers and malignant frac- for the primary bone marrow sample,see Supplementary Informa- tions of clones in each primary tumour and relapse sample.Similar to tion),we were able to calculate the fraction of total malignant cells in UPN 933124,multiple mutation clusters(2-4)were present in each of each clone.Clone 1 is the founding clone;12.74%of the tumour cells the primary tumours from four patients (UPN 869586,UPN 426980, contain only this set of mutations.Clones 2,3 and 4 evolved from clone UPN 452198 and UPN 758168).However,only one major cluster was 1.The additional mutations in clones 2 and 3 may have provided a detected in each of the primary tumours from the three other patients growth or survival advantage,as 53.12%and 29.04%of the tumour (UPN 804168,UPN 573988 and UPN 400220)(Fig.1c and Sup- cells belonged to these clones,respectively.Only 5.10%of the tumour plementary Table 10).Importantly,all eight patients gained relapse- cells were from clone 4,indicating that it may have arisen last(Fig.2a). specific mutations,although the number of clusters in the relapse However,the relapse clone evolved from clone 4.A single clone samples varied (Fig.1). a Clonal fractions at initial diagnosis Day 170 First relapse 12.74%● 29.04% HSCs 5.10% *DNMT3A.NPMI.FLT3.PTPRT.SMC3 Y1-C. ● 5312%@ AML1/UPN933124 Cell type: Mutatio ●Normal●AML se sp Pathogenic mutations Pnmary spe Model1UPNs400220.573988,804168) 40 20 Chemotherapy 20406080100 Tumour varlant (36) Model2UPNs426980,452198.758168.869586,933124 AMLL43/UPN869586 60 0 80100 Chematherapy Figure 2 Graphical representation of clonal evolution from the primary two major patterns of tumour evolution in AML Model 1 shows the dominant tumour to relapse in UPN 933124,and patterns of tumour evolution clone in the primary tumour evolving into the relapse clone by gaining relapse- observed in eight primary tumour and relapse pairs.a,The founding clone in specific mutations;this pattern was identified in three primary tumour and the primary tumour in UPN 933124 contained somatic mutations in relapse pairs(UPN 804168,UPN 573988 and UPN 400220).Model 2 shows a DNMT3A,NPMI,PTPRT,SMC3 and FLT3 that are all recurrent in AML and minor clone carrying the vast majority of the primary tumour mutations probably relevant for pathogenesis;one subclone within the founding clone survived and expanded at relapse.This pattern was observed in five primary evolved to become the dominant clone at relapse by acquiring additional tumour and relapse pairs (UPN 933124,UPN 452198,UPN 758168,UPN mutations,including recurrent mutations in ETV6 and MYO18B,and a 426980 and UPN869586. WNKI-WAC fusion gene.HSC,haematopoietic stem cell.b,Examples of the 508 NATURE I VOL 481 26 JANUARY 2012 2012 Macmillan Publishers Limited.All rights reserved
be present in virtually all the tumour cells at presentation and at relapse, as the variant frequency of these mutations is ,40–50%. Clone 2 (with cluster 2 mutations) and clone 3 (with cluster 3 mutations) must be derived from clone 1, because virtually all the cells in the sample contain the cluster 1 mutations (Fig. 2a). It is likely that a single cellfrom clone 3 gained a set of mutations (cluster 4) to form clone 4: these survived chemotherapy and evolved to become the dominant clone at relapse. We do not know whether any of the cluster 4 mutations conferred chemotherapy resistance; although none had translational consequences, we cannot rule out a relevant regulatory mutation in this cluster. Assuming that all the mutations detected are heterozygous in the primary tumour sample (with a malignant cellular content at 93.72% for the primary bone marrow sample, see Supplementary Information), we were able to calculate the fraction of total malignant cells in each clone. Clone 1 is the founding clone; 12.74% of the tumour cells contain only this set of mutations. Clones 2, 3 and 4 evolved from clone 1. The additional mutations in clones 2 and 3 may have provided a growth or survival advantage, as 53.12% and 29.04% of the tumour cells belonged to these clones, respectively. Only 5.10% of the tumour cells were from clone 4, indicating that it may have arisen last (Fig. 2a). However, the relapse clone evolved from clone 4. A single clone containing all of the cluster 5 mutations was detected in the relapse sample; clone 5 evolved from clone 4, but gained 78 new somatic alterations after sampling at day 170. As all mutations in clone 5 appear to be present in all relapse tumour cells, we suspect that one or more of the mutations in this clone provided a strong selective advantage that contributed to relapse. The ETV6 mutation, the MYO18B mutation, and/or the WNK1-WAC fusion are the most likely candidates, as ETV6, MYO18B and WAC are recurrently mutated in AML. We evaluated the mutation clusters in the seven additional primary tumour–relapse pairs by assessing peaks of allele frequency using kernel density estimation (Supplementary Fig. 11 and Supplementary Information). We thus inferred the numbers and malignant fractions of clones in each primary tumour and relapse sample. Similar to UPN 933124, multiple mutation clusters (2–4) were present in each of the primary tumours from four patients (UPN 869586, UPN 426980, UPN 452198 and UPN 758168). However, only one major cluster was detected in each of the primary tumours from the three other patients (UPN 804168, UPN 573988 and UPN 400220) (Fig. 1c and Supplementary Table 10). Importantly, all eight patients gained relapsespecific mutations, although the number of clusters in the relapse samples varied (Fig. 1). Cell type: Mutations: Relapse specific (cluster 5) Primary specific (cluster 2) Relapse enriched (cluster 4) Founding (cluster 1) Relapse enriched (cluster 3) Normal AML Pathogenic mutations Random mutations in HSCs Clonal fractions at initial diagnosis Day 170 First relapse AML1/UPN933124 DNMT3A, NPM1, FLT3, PTPRT, SMC3 ETV6, WNK1-WAC, MYO18B HSCs a b 12.74% 29.04% 5.10% 53.12% Chemotherapy Model 2 (UPNs 426980, 452198, 758168, 869586, 933124) Model 1 (UPNs 400220, 573988, 804168) Tumour variant frequency (%) Relapse variant frequency (%) 0 20 40 60 80 100 20 40 60 80 100 AML AML40/UPN804168 40 / UPN804168 Tumour variant frequency (%) Relapse variant frequency (%) 0 20 40 60 80 100 20 40 60 80 100 AML AML43/UPN869586 43 / UPN869586 Chemotherapy Chemotherapy Figure 2 | Graphical representation of clonal evolution from the primary tumour to relapse in UPN 933124, and patterns of tumour evolution observed in eight primary tumour and relapse pairs. a, The founding clone in the primary tumour in UPN 933124 contained somatic mutations in DNMT3A, NPM1, PTPRT, SMC3 and FLT3 that are all recurrent in AML and probably relevant for pathogenesis; one subclone within the founding clone evolved to become the dominant clone at relapse by acquiring additional mutations, including recurrent mutations in ETV6 and MYO18B, and a WNK1-WAC fusion gene. HSC, haematopoietic stem cell. b, Examples of the two major patterns of tumour evolution in AML. Model 1 shows the dominant clone in the primary tumour evolving into the relapse clone by gaining relapsespecific mutations; this pattern was identified in three primary tumour and relapse pairs (UPN 804168, UPN 573988 and UPN 400220). Model 2 shows a minor clone carrying the vast majority of the primary tumour mutations survived and expanded at relapse. This pattern was observed in five primary tumour and relapse pairs (UPN 933124, UPN 452198, UPN 758168, UPN 426980 and UPN 869586). RESEARCH LETTER 508 | NATURE | VOL 481 | 26 JANUARY 2012 ©2012 Macmillan Publishers Limited. All rights reserved
ETTER RESEARCH Two major patterns of clonal evolution were detected at relapse examined the 456 relapse-specific mutations and 3,590 primary tumour (Fig.2band Supplementary Fig.3):in cases with pattern 1,the dominant point mutations from all eight cases as a group,and found that the clone in the primary tumour gained additional mutations and evolved transversion frequency is significantly higher for relapse-specific into the relapse clone(UPN 804168,UPN 573988 and UPN 400220). mutations (46%)than for primary tumour mutations (30.7%) These patients may simply be inadequately treated(for example,elderly (P=3.71X 101),indicating that chemotherapy has a substantial patients who cannot tolerate aggressive consolidation,like UPN effect on the mutational spectrum at relapse.Similar results were 573988),or they may have mutations in their founding clones(or obtained when we limited the analysis to the 213 mutations that had germline variants)that make these cells more resistant to therapy 0%variant frequency in the primary tumour samples(Supplementary (UPN 804168 and UPN 400220).In patients with pattern 2,a minor Fig.Ib);the transversion frequency for relapse-specific mutations was subclone carrying the vast majority (but not all)of the primary tumour 50.4%,versus 31.4%for primary tumour samples (P=3.89 X 10). mutations survived,gained mutations,and expanded at relapse;a sub Very few copy-number alterations were detected in the eight relapse set of primary tumour mutations was often eradicated by therapy,and samples,suggesting that the increased transversion rate is not asso- were not detected at relapse(UPN 758168,UPN 933124,UPN 452198. ciated with generalized genomic instability(Supplementary Fig.12). UPN 426980 and UPN 869586).Specific mutations in a key subclone We first described the use of deep sequencing to define precisely the may contribute to chemotherapy resistance,or the mutations import- variant allele frequencies of the mutations in the AML genome of case ant for relapse may be acquired during tumour evolution,or both. 933124(ref.3),and here have refined and extended this technique to Notably,in cases 426980 and 758168,a second primary tumour clone examine clonal evolution at relapse.The analysis of eight primary survived chemotherapy and was also present at relapse(Fig.Ic and AMLand relapse pairs has revealed unequivocal evidence for a common Supplementary Fig.3).Owing to current technical limits in our ability origin of tumour subpopulations;a dominant mutation cluster repre- to detect mutations in rare cells(mostly related to currently achievable senting the founding clone was discovered in the primary tumour levels of coverage with whole genome sequencing),our models repres- sample in all cases.The relationship of the founding clone (and sub- ent a minimal estimate of the clonal heterogeneity in AML. clones thereof)to the 'leukaemia initiating cell'is not yet clear- All eight patients received cytarabine and anthracycline for induc- purification of clonal populations and functional testing would be tion therapy,and additional cytotoxic chemotherapy for consolidation; required to establish this relationship.We observed the loss of primary treatment histories are summarized in Supplementary Table 2 and tumour subclones at relapse in four ofeight cases,suggesting that some described in Supplementary Information.To investigate the potential subclones are indeed eradicated by therapy(Figs 1 and 2 and Sup- impact of treatment on relapse mutation types,we compared the six plementary Fig.3).Some mutations gained at relapse may alter the classes of transition and transversion mutations in the primary tumour growth properties of AML cells,or confer resistance to additional with the relapse-specific mutations in all eight patients (Fig.3a). chemotherapy.Regardless,each tumour displayed clear evidence of Although C.G>T.A transitions are the most common mutations clonal evolution at relapse and a higher frequency of transversions that found in both primary and relapse AML genomes,their frequencies were probably induced by DNA damage from chemotherapy. are significantly different between the primary tumour mutations Although chemotherapy is required to induce initial remissions in (51.1%)and relapse-specific mutations (40.5%)(P=2.99 X 10-7). AML patients,our data also raise the possibility that it contributes to Moreover,we observed an average of 4.5%,5.3%and 4.2%increase relapse by generating new mutations in the founding clone or one ofits inAT-→CG(P=9.13×10),CG→AT(P=0.00312)and subclones,which then can undergo selection and clonal expansion. C.GG.C (P=0.00366)transversions,respectively,in relapse- These data demonstrate the critical need to identify the disease-causing specific mutations.Notably,an increased A.TC.G transversion rate mutations for AML,so that targeted therapies can be developed that has also been observed in cases of chronic lymphocytic leukaemia with avoid the use of cytotoxic drugs,many of which are mutagens. mutated immunoglobulin genes.C.GA.T transversions are the This study extends the findings of previous studiest-2,which most common mutation in lung cancer patients who were exposed to recently described patterns of clonal evolution in ALL patients using tobacco-borne carcinogens's(Fig.3band Supplementary Table 11).We fluorescence in situ hybridization and/or copy number alterations detected by SNP arrays,and it enhances the understanding of genetic 6 changes acquired during disease progression,as previously described 100 for breast and pancreatic cancer metastases-.Our data provide complementary information on clonal evolution in AML,using a much larger set of mutations that were quantified with deep sequen- 80 90 cing;this provides an unprecedented number of events that can be used to define precisely clonal size and mutational evolution at relapse. 60 Both ALL and AML share common features of clonal heterogeneity at presentation followed by dynamic clonal evolution at relapse,includ- ing the addition of new mutations that may be relevant for relapse pathogenesis.Clonal evolution can also occur after allogeneic trans- plantation (for example,loss of mismatched HLA alleles via a uni- parental disomy mechanism),demonstrating that the type of therapy itself can affect clonal evolution at relapse2.Taken together,these data demonstrate that AML cells routinely acquire a small number of A-C A A.T C.A C.G Primary tumour Relapse Pw0157 P-3.71x10-1 additional mutations at relapse,and suggest that some of these muta- P0003 P200 tions may contribute to clonal selection and chemotherapy resistance. The AML genome in an individual patient is clearly a 'moving target'; Figure 3 Comparison of mutational classes between primary tumours and eradication of the founding clone and all of its subclones will be relapse samples.a,Fraction of the primary tumour and relapse-specific required to achieve cures. mutations in each of the transition and transversion categories.b,Transversion frequencies of the primary tumour and relapse-specific mutations from eight METHODS SUMMARY AML tumour and relapse pairs.456 relapse-specific mutations and 3,590 Illumina paired-end reads were aligned to NCBI build36 using BWA 0.5.5(http:// primary tumour mutations from eight cases were used for assessing statistical sourceforge.net/projects/bio-bwa/).Somatic mutations were identified using significance using proportion tests. SomaticSniper and a modified version of the SAMtools indel caller.Structural 26 JANUARY 2012 VOL 481 NATURE 509 2012 Macmillan Publishers Limited.All rights reserved
Two major patterns of clonal evolution were detected at relapse (Fig. 2b and Supplementary Fig. 3): in caseswith pattern 1, the dominant clone in the primary tumour gained additional mutations and evolved into the relapse clone (UPN 804168, UPN 573988 and UPN 400220). These patients may simply be inadequately treated (for example, elderly patients who cannot tolerate aggressive consolidation, like UPN 573988), or they may have mutations in their founding clones (or germline variants) that make these cells more resistant to therapy (UPN 804168 and UPN 400220). In patients with pattern 2, a minor subclone carrying the vast majority (but not all) of the primary tumour mutations survived, gained mutations, and expanded at relapse; a subset of primary tumour mutations was often eradicated by therapy, and were not detected at relapse (UPN 758168, UPN 933124, UPN 452198, UPN 426980 and UPN 869586). Specific mutations in a key subclone may contribute to chemotherapy resistance, or the mutations important for relapse may be acquired during tumour evolution, or both. Notably, in cases 426980 and 758168, a second primary tumour clone survived chemotherapy and was also present at relapse (Fig. 1c and Supplementary Fig. 3). Owing to current technical limits in our ability to detect mutations in rare cells (mostly related to currently achievable levels of coverage with whole genome sequencing), our models represent a minimal estimate of the clonal heterogeneity in AML. All eight patients received cytarabine and anthracycline for induction therapy, and additional cytotoxic chemotherapy for consolidation; treatment histories are summarized in Supplementary Table 2 and described in Supplementary Information. To investigate the potential impact of treatment on relapse mutation types, we compared the six classes of transition and transversion mutations in the primary tumour with the relapse-specific mutations in all eight patients (Fig. 3a). Although CNGRTNA transitions are the most common mutations found in both primary and relapse AML genomes, their frequencies are significantly different between the primary tumour mutations (51.1%) and relapse-specific mutations (40.5%) (P 5 2.993 1027 ). Moreover, we observed an average of 4.5%, 5.3% and 4.2% increase in ANTRCNG (P 5 9.1331027 ), CNGRANT (P 5 0.00312) and CNGRGNC (P 5 0.00366) transversions, respectively, in relapsespecific mutations. Notably, an increased ANTRCNG transversion rate has also been observed in cases of chronic lymphocytic leukaemia with mutated immunoglobulin genes17. CNGRANT transversions are the most common mutation in lung cancer patients who were exposed to tobacco-borne carcinogens18 (Fig. 3b and Supplementary Table 11).We examined the 456 relapse-specific mutations and 3,590 primary tumour point mutations from all eight cases as a group, and found that the transversion frequency is significantly higher for relapse-specific mutations (46%) than for primary tumour mutations (30.7%) (P 5 3.713 10211), indicating that chemotherapy has a substantial effect on the mutational spectrum at relapse. Similar results were obtained when we limited the analysis to the 213 mutations that had 0% variant frequency in the primary tumour samples (Supplementary Fig. 1b); the transversion frequency for relapse-specific mutations was 50.4%, versus 31.4% for primary tumour samples (P 5 3.893 1029 ). Very few copy-number alterations were detected in the eight relapse samples, suggesting that the increased transversion rate is not associated with generalized genomic instability (Supplementary Fig. 12). We first described the use of deep sequencing to define precisely the variant allele frequencies of the mutations in the AML genome of case 933124 (ref. 3), and here have refined and extended this technique to examine clonal evolution at relapse. The analysis of eight primary AML and relapse pairs has revealed unequivocal evidence for a common origin of tumour subpopulations; a dominant mutation cluster representing the founding clone was discovered in the primary tumour sample in all cases. The relationship of the founding clone (and subclones thereof) to the ‘leukaemia initiating cell’ is not yet clear— purification of clonal populations and functional testing would be required to establish this relationship. We observed the loss of primary tumour subclones at relapse in four of eight cases, suggesting that some subclones are indeed eradicated by therapy (Figs 1 and 2 and Supplementary Fig. 3). Some mutations gained at relapse may alter the growth properties of AML cells, or confer resistance to additional chemotherapy. Regardless, each tumour displayed clear evidence of clonal evolution at relapse and a higher frequency of transversions that were probably induced by DNA damage from chemotherapy. Although chemotherapy is required to induce initial remissions in AML patients, our data also raise the possibility that it contributes to relapse by generating new mutations in the founding clone or one of its subclones, which then can undergo selection and clonal expansion. These data demonstrate the critical need to identify the disease-causing mutations for AML, so that targeted therapies can be developed that avoid the use of cytotoxic drugs, many of which are mutagens. This study extends the findings of previous studies19–21, which recently described patterns of clonal evolution in ALL patients using fluorescence in situ hybridization and/or copy number alterations detected by SNP arrays, and it enhances the understanding of genetic changes acquired during disease progression, as previously described for breast and pancreatic cancer metastases22–25. Our data provide complementary information on clonal evolution in AML, using a much larger set of mutations that were quantified with deep sequencing; this provides an unprecedented number of events that can be used to define precisely clonal size and mutational evolution at relapse. Both ALL and AML share common features of clonal heterogeneity at presentation followed by dynamic clonal evolution at relapse, including the addition of new mutations that may be relevant for relapse pathogenesis. Clonal evolution can also occur after allogeneic transplantation (for example, loss of mismatched HLA alleles via a uniparental disomy mechanism), demonstrating that the type of therapy itself can affect clonal evolution at relapse26,27. Taken together, these data demonstrate that AML cells routinely acquire a small number of additional mutations at relapse, and suggest that some of these mutations may contribute to clonal selection and chemotherapy resistance. The AML genome in an individual patient is clearly a ‘moving target’; eradication of the founding clone and all of its subclones will be required to achieve cures. METHODS SUMMARY Illumina paired-end reads were aligned to NCBI build36 using BWA 0.5.5 (http:// sourceforge.net/projects/bio-bwa/). Somatic mutations were identified using SomaticSniper28 and a modified version of the SAMtools indel caller. Structural Transversion frequency (%) 0 20 40 60 80 100 Primary tumour Relapse tumour Primary tumour Relapse tumour b Primary tumour Relapse P = 3.71 × 10–11 Fraction of mutations (%) 0 20 40 60 80 100 a P = 9.13 × 10–7 A C P = 0.1573 A G P = 0.3456 A T P = 0.003122 C A P = 0.003664 C G P = 2.99 × 10–7 C T Figure 3 | Comparison of mutational classes between primary tumours and relapse samples. a, Fraction of the primary tumour and relapse-specific mutations in each of the transition and transversion categories. b, Transversion frequencies of the primary tumour and relapse-specific mutations from eight AML tumour and relapse pairs. 456 relapse-specific mutations and 3,590 primary tumour mutations from eight cases were used for assessing statistical significance using proportion tests. LETTER RESEARCH 26 JANUARY 2012 | VOL 481 | NATURE | 509 ©2012 Macmillan Publishers Limited. All rights reserved
RESEARCH LETTER variations were identified using BreakDancer.All predicted non-repetitive so- 18.Ding,L.et al.Somatic mutations affect key pathways in lung adenocarcinoma. matic SNVs,indels and all structural variants were included on custom sequence re455,1069-1075(2008) capture arrays from Roche Nimblegen.Illumina 2 X 100-bp paired-end sequen- 19.Mullighan,C.G.et al.Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia.Science 322,1377-1380(2008). cing reads were produced after elution from capture arrays.VarScan and a read 20.Anderson,K.etal Genetic variegation of clonal architecture and propagating cells remapping strategy using Crossmatch(P.Green,unpublished data)and BWA in leukaemia.Nature 469.356-361 (2011). were used for determining the validation status of predicted SNVs,indels and 21.Notta,F.et al.Evolution of human BCR-ABL1 lymphoblastic leukaemia-initiating structural variants.A complete description of the materials and methods is pro- cells.Nature469.362-367(20111. vided in Supplementary Information.All sequence variants for the AML tumour 22. Ding,L.et al.Genome remodelling in a basal-like breast cancer metastasis and xenograft.Nature 464,999-1005(2010) samples from eight cases have been submitted to dbGaP (accession number 23. Shah,S.P.et al.Mutational evolution in a lobular breast tumour profiled at single phs000I59.v4.p2). nucleotide resolution.Nature 461,809-813(2009). 24.Yachida,S.et al Distant metastasis ccurs late during the genetic evolution of Received 29 March;accepted 29 November 2011 pancreatic cancer.Nature 467,1114-1117 (2010). Published online 11 January 2012. Navin,N.et al.Tumour evolution inferred by single-cell sequencing.Nature 472, 90-942011). 1.Testa,J.R.,Mintz,U.,Rowley,J.D.Vardiman,J.W.Golomb,H.M.Evolution of 26.Vago,L.etal Loss of mism natched HLA in leukemia after stem-cell transplantation karyotypes in acute nonlymphocytic leukemia.Cancer Res.39,3619-3627 N.Engl.1Med.361,478-488(2009) (1979) 27.Villalobos,I.B.et al.Relapse of leukemia with loss of mismatched HLA resulting 2 Garson,O.M.et al.Cytogenetic studies of 103 patients with acute myelogenous from uniparental disomy after haploidentical hematopoietic stem cell transplantation.Blood 115,3158-3161 (2010). leukemia in relapse.Cancer Genet Cytogenet 40,187-202(1989). 38 3. Ley,T.J.et al.DNA sequencing of a cytogenetically normal acute myeloid Larson,D.E.et al.SomaticSniper:identification of somatic point mutations in leukaemia genome.Nature 456,66-72 (2008). whole genome sequencing data.Bioinformatics (in the press). 4. Li,H.et al.The Sequence Alignment/Map format and SAMtools.Bioinformatics 25, Supplementary Information is linked to the online version of the paper at 2078-2079(2009). www.nature.com/nature. 5. Chen,K.et al BreakDancer:an algorithm for high-resolution mapping of genomic structural variation.Nature Methods 6,677-681 (2009). Acknowledgements We thank the Analysis Pipeline group for developing the 6. Koboldt,D.C.et al.VarScan:variant detection in massively parallel sequencingo automated sequence analysis pipelines;the LIMS group for developing tools and individual and pooled samples.Bioinformatics 25,2283-2285(2009). software to manage samples and sequencing:the Systems group for providing the IT 7. Mardis,ER.et al Recurring mutations found by sequencing an acute myeloid infrastructure and HPC solutions required for sequencing and analysis;and leukemia genome.N.Engl.J.Med.361,1058-1066(2009). R.T.Demeter for experimental support We also thank The Cancer Genome Atlas for 8. Ley,T.J.etal.DNMT3A mutations in acute myeloid leukemia.N.Engl.J.Med.363, allowing us to use unpublished data for this study,and the Washington University 2424-2433(2010). Cancer Genome Initiative for their support.This work was funded by grants to R.K.W. 9. Nakao,M.etal Inte of the flt3 gene found in acute myeloid and the National Human Genome Research Institute(NHGRI U54 HG003079),and leukemia.Leukemia 10,1911-1918(1996). grants to T.J.L from the National Cancer Institute(PO1 CA101937)and the 10.Falini,B.etal.Cytoplasmic nucleophosmin in acute myelogenous leukemia with a Barnes-Jewish Hospital Foundation(00335-0505-02). normal karyotype.N.Engl.J.Med.352,254-266(2005). Author Contributions TJ.L,L.D..J.F.D.ER.M.and R.K.W.designed the experiments. 11.Ward,P.S.et al.The common feature of leukemia-associated IDH1 and IDH2 LD.and TJ.L.led dataanalysis.LD.D.EL.CA.M..D.C.K..J.S.W.M.D.M..J.W.W.C.L..D.S. mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.Cancer Cell 17,225-234 (2010). C.C.H.K.C.H.S.,J.K.-V,M.C.W.M.AW.W.D.S.,J.E.P.and S.K.performed data analysis J.F.M.,M.D.M.and L.D.prepared figuresandtables.J.S.W..J.K.R.,M.AY.,T.L.R.S.F.LLF. 12.King-Underwood,L,Renshaw,J.Pritchard-Jones,K.Mutations in the Wilms VJ.M.,LS.,LC.S.D.M.and T.L.V.performed laboratory experiments.S.H.and P.W. tumor gene WT1 in leukemias.Blood 87,2171-2179(1996). provided samples and clinical data.DJ.D.provided informatics support TJ.L.D.C.L. 13.Gao,J.et al.Isolation of a yeast artificial chromosome spanning the 8:21 M.H.T.E.R.M.R.K.W.and J.F.D.developed projectconcept.LD..TJ.L.MJ.W.TA.G.and translocation breakpoint t(8:21)(q22:q22.3)in acute myelogenous leukemia. Proc.Natl Acad.Sci USA 88.4882-4886 (1991) J.F.D.wrote the manuscript. 14.Kirito,K.etal A novel RUNX1 mutation in familial platelet disorder with propensity Author Information All sequence variants for the AML tumour samples from eight to develop myeloid malignancies.Haematologica 93,155-156(2008). cases have been submitted to dbGaP under ac ession number phs000159.v4.p2. 15.Van Vlierberghe,P.et al.PHF6 mutations in adult acute myeloid leukemia. Reprints and permissions information is available at www.nature.com/reprints.This Leukemia25,130-134(2011). paper is distributed under the terms of the Creative Commons 16.Barjesteh van Waalwijk van Doorn-Khosrovani,S.et al.Somatic heterozygous Attribution-Non-Commercial-Share Alike licence,and is freely available to all readers at mutations in ETV6 (TEL)and frequent absence of ETV6 protein in acute myeloid www.nature.com/nature.The authors declare no competing financial interests. 1 eukemia..0 ncogene24,4129-4137(2005). Readers are welcome to comment on the online version of this article at 17.Puente.X.S.et al.Whole-genome sequencing identifies recurrent mutations in www.nature.com/nature.Correspondence and requests for materials should be chronic lymphocytic leukaemia.Nature 475,101-105(2011). addressed to T_J.L (timley@wustledu). 510 I NATURE VOL 481 26 JANUARY 2012 2012 Macmillan Publishers Limited.All rights reserved
variations were identified using BreakDancer5 . All predicted non-repetitive somatic SNVs, indels and all structural variants were included on custom sequence capture arrays from Roche Nimblegen. Illumina 2 3 100-bp paired-end sequencing reads were produced after elution from capture arrays. VarScan6 and a read remapping strategy using Crossmatch (P. Green, unpublished data) and BWA were used for determining the validation status of predicted SNVs, indels and structural variants. A complete description of the materials and methods is provided in Supplementary Information. All sequence variants for the AML tumour samples from eight cases have been submitted to dbGaP (accession number phs000159.v4.p2). Received 29 March; accepted 29 November 2011. Published online 11 January 2012. 1. Testa, J. R., Mintz, U., Rowley, J. D., Vardiman, J. W. & Golomb, H. M. Evolution of karyotypes in acute nonlymphocytic leukemia. Cancer Res. 39, 3619–3627 (1979). 2. Garson, O. M. et al. Cytogenetic studies of 103 patients with acute myelogenous leukemia in relapse. Cancer Genet. Cytogenet. 40, 187–202 (1989). 3. Ley, T. J. et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 456, 66–72 (2008). 4. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009). 5. Chen, K. et al. BreakDancer: an algorithm for high-resolution mapping of genomic structural variation. Nature Methods 6, 677–681 (2009). 6. Koboldt, D. C. et al. VarScan: variant detection in massively parallel sequencing of individual and pooled samples. Bioinformatics 25, 2283–2285 (2009). 7. Mardis, E. R. et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N. Engl. J. Med. 361, 1058–1066 (2009). 8. Ley, T. J. et al. DNMT3A mutations in acute myeloid leukemia. N. Engl. J. Med. 363, 2424–2433 (2010). 9. Nakao, M. et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 10, 1911–1918 (1996). 10. Falini, B. et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N. Engl. J. Med. 352, 254–266 (2005). 11. Ward, P. S. et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 17, 225–234 (2010). 12. King-Underwood, L., Renshaw, J. & Pritchard-Jones, K. Mutations in the Wilms’ tumor gene WT1 in leukemias. Blood 87, 2171–2179 (1996). 13. Gao, J. et al. Isolation of a yeast artificial chromosome spanning the 8;21 translocation breakpoint t(8;21)(q22;q22.3) in acute myelogenous leukemia. Proc. Natl Acad. Sci. USA 88, 4882–4886 (1991). 14. Kirito, K. et al. A novel RUNX1 mutation in familial platelet disorder with propensity to develop myeloid malignancies. Haematologica 93, 155–156 (2008). 15. Van Vlierberghe, P. et al. PHF6 mutations in adult acute myeloid leukemia. Leukemia 25, 130–134 (2011). 16. Barjesteh van Waalwijk van Doorn-Khosrovani, S. et al. Somatic heterozygous mutations in ETV6 (TEL) and frequent absence of ETV6 protein in acute myeloid leukemia. Oncogene 24, 4129–4137 (2005). 17. Puente, X. S. et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature 475, 101–105 (2011). 18. Ding, L. et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455, 1069–1075 (2008). 19. Mullighan, C. G. et al. Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia. Science 322, 1377–1380 (2008). 20. Anderson, K. et al. Genetic variegation of clonal architecture and propagating cells in leukaemia. Nature 469, 356–361 (2011). 21. Notta, F. et al. Evolution of human BCR-ABL1 lymphoblastic leukaemia-initiating cells. Nature 469, 362–367 (2011). 22. Ding, L. et al. Genome remodelling in a basal-like breast cancer metastasis and xenograft. Nature 464, 999–1005 (2010). 23. Shah, S. P. et al. 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Acknowledgements We thank the Analysis Pipeline group for developing the automated sequence analysis pipelines; the LIMS group for developing tools and software to manage samples and sequencing; the Systems group for providing the IT infrastructure and HPC solutions required for sequencing and analysis; and R. T. Demeter for experimental support. We also thank The Cancer Genome Atlas for allowing us to use unpublished data for this study, and the Washington University Cancer Genome Initiative for their support. This work was funded by grants to R.K.W. and the National Human Genome Research Institute (NHGRI U54 HG003079), and grants to T.J.L. from the National Cancer Institute (PO1 CA101937) and the Barnes-Jewish Hospital Foundation (00335-0505-02). Author Contributions T.J.L., L.D., J.F.D., E.R.M. and R.K.W. designed the experiments. L.D. and T.J.L. led data analysis. L.D., D.E.L., C.A.M., D.C.K., J.S.W., M.D.M., J.W.W., C.L., D.S., C.C.H., K.C., H.S., J.K.-V., M.C.W., M.A.W., W.D.S., J.E.P. and S.K. performed data analysis. J.F.M.,M.D.M. and L.D. prepared figures and tables. J.S.W., J.K.R., M.A.Y., T.L., R.S.F., L.L.F., V.J.M., L.S., L.C., S.D.M. and T.L.V. performed laboratory experiments. S.H. and P.W. provided samples and clinical data. D.J.D. provided informatics support. T.J.L., D.C.L., M.H.T., E.R.M., R.K.W. and J.F.D. developed project concept. L.D., T.J.L., M.J.W., T.A.G. and J.F.D. wrote the manuscript. Author Information All sequence variants for the AML tumour samples from eight cases have been submitted to dbGaP under accession number phs000159.v4.p2. Reprints and permissions information is available at www.nature.com/reprints. This paper is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence, and is freely available to all readers at www.nature.com/nature. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. Correspondence and requests for materials should be addressed to T.J.L. (timley@wustl.edu). RESEARCH LETTER 510 | NATURE | VOL 481 | 26 JANUARY 2012 ©2012 Macmillan Publishers Limited. All rights reserved
