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 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 level1,2. 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, DDX41 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 mm23 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 sample3, 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, PDXDC1 E421K in transversions, probably due to DNA damage caused by cytotoxic and TTN E14263K) (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), NPM1 (ref. 10), IDH1 (ref. 7), IDH2 (ref. 11), WT1 (ref. 12), to determine whether clonal evolution contributes to relapse, we per- RUNX1 (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 from eight 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 reported3. 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 .253 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 of high-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 identified4,5 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 specimens6 (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– 1 to 4 were 46.86%, 24.89%, 16.00% and 2.39%, respectively (Fig. 1b and 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 1 mutations; sequenced case (UPN 933124). A total of 413 somatic events from tiers assuming that nearly all of these mutations are heterozygous, they must 1The Genome Institute, Washington University, St Louis, Missouri 63108, USA. 2Department of Genetics, Washington University, St Louis, Missouri 63110, USA. 3Department of Internal Medicine, Division of Oncology, Washington University, St Louis, Missouri 63110, USA. 4Siteman Cancer Center, Washington University, St Louis, Missouri 63110, USA. 5Department of Pathology and Immunology, Washington University, St Louis, Missouri 63110, USA. 6Division 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 a b 1,400 Relapse specific 100 1,292 Cluster ID Primary tumour specific AMLAML1/UPN9331241 / UPN933124 1 2 1,200 Shared 3 80 4 5 1,000 Outlier 882 60 800 100 658 (%) allele frequency Variant PTPRT (P1232L) SMC3 (G662C) AML1/UPN933124 MYO18B (A2317T) 600 80 40 496 TTN (E14263K) ETV6 (R105P) NPM1 412 (W287fs) 60 Number of mutations 400 FLT3 (594in_frame_ins) DNMT3A (L723fs) 307 40 20 200 150 (%) Relapse variant allele frequency 118 20 0 0 0 0 20 40 60 80 100 Primary Relapse 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 Primary tumour variant allele frequency (%) tumour 84.5% purity 93.7% purity 452198 573988 400220 933124 758168 804168 426980 869586 Cases c AML43/UPN869586 AML28/UPN426980 AML31/UPN452198 AML15/UPN758168 60 Cluster 60 Cluster 60 Cluster 60 Cluster 1 1 1 1 40 2 40 2 40 2 40 2 3 3 3 3 20 4 20 4 20 4 20 Variant allele Variant Variant allele Variant Variant allele Variant Variant allele Variant frequency (%) frequency frequency (%) frequency frequency (%) frequency frequency (%) frequency 5 0 0 0 0 Primary tumour Relapse Primary tumour Relapse Primary tumour Relapse Primary tumour Relapse 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/UPN573988 AML27/UPN400220 60 Cluster 60 Cluster 60 Cluster 1 1 1 40 2 40 2 40 2 20 20 20 Variant allele Variant Variant allele Variant Variant allele Variant frequency (%) frequency frequency (%) frequency frequency (%) frequency 0 0 0 Primary tumour Relapse Primary tumour Relapse Primary 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.