T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`original article
`
`Recurring Mutations Found by Sequencing
`an Acute Myeloid Leukemia Genome
`Elaine R. Mardis, Ph.D., Li Ding, Ph.D., David J. Dooling, Ph.D.,
`David E. Larson, Ph.D., Michael D. McLellan, B.S., Ken Chen, Ph.D.,
`Daniel C. Koboldt, M.S., Robert S. Fulton, M.S., Kim D. Delehaunty, B.A.,
`Sean D. McGrath, M.S., Lucinda A. Fulton, M.S., Devin P. Locke, Ph.D.,
`Vincent J. Magrini, Ph.D., Rachel M. Abbott, B.S., Tammi L. Vickery, B.S.,
`Jerry S. Reed, M.S., Jody S. Robinson, M.S., Todd Wylie, B.S., Scott M. Smith,
`Lynn Carmichael, B.S., James M. Eldred, Christopher C. Harris, B.S.,
`Jason Walker, B.A., B.S., Joshua B. Peck, M.B.A., Feiyu Du, M.S.,
`Adam F. Dukes, B.A., Gabriel E. Sanderson, B.S., Anthony M. Brummett,
`Eric Clark, Joshua F. McMichael, B.S., Rick J. Meyer, M.S.,
`Jonathan K. Schindler, B.S., B.A., Craig S. Pohl, M.S., John W. Wallis, Ph.D.,
`Xiaoqi Shi, M.S., Ling Lin, M.S., Heather Schmidt, B.S., Yuzhu Tang, M.D.,
`Carrie Haipek, M.S., Madeline E. Wiechert, M.S., Jolynda V. Ivy, M.B.A.,
`Joelle Kalicki, B.S., Glendoria Elliott, Rhonda E. Ries, M.A.,
`Jacqueline E. Payton, M.D., Ph.D., Peter Westervelt, M.D., Ph.D.,
`Michael H. Tomasson, M.D., Mark A. Watson, M.D., Ph.D., Jack Baty, B.A.,
`Sharon Heath, William D. Shannon, Ph.D., Rakesh Nagarajan, M.D., Ph.D.,
`Daniel C. Link, M.D., Matthew J. Walter, M.D., Timothy A. Graubert, M.D.,
`John F. DiPersio, M.D., Ph.D., Richard K. Wilson, Ph.D., and Timothy J. Ley, M.D.
`
`A bs tr ac t
`
`From the Departments of Genetics (E.R.M.,
`L.D., V.J.M., R.K.W., T.J.L.), Medicine
`(R.E.R., P.W., M.H.T., S.H., W.D.S., D.C.L.,
`M.J.W., T.A.G., J.F.D., T.J.L.), and Pathology
`and Immunology (J.E.P., M.A.W., R.N.);
`the Genome Center (E.R.M., L.D., D.J.D.,
`D.E.L., M.D.M., K.C., D.C.K., R.S.F., K.D.D.,
`S.D.M., L.A.F., D.P.L., V.J.M., R.M.A.,
`T.L.V., J.S. Reed, J.S. Robinson, T.W., S.M.S.,
`L.C., J.M.E., C.C.H., J.W., J.B.P., F.D.,
`A.F.D., G.E.S., A.M.B., E.C., J.F.M., R.J.M.,
`J.K.S., C.S.P., J.W.W., X.S., L.L., H.S., Y.T.,
`C.H., M.E.W., J.V.I., J.K., G.E., M.A.W.,
`R.K.W., T.J.L.); Siteman Cancer Center
`(P.W., M.H.T., M.A.W., S.H., W.D.S., R.N.,
`D.C.L., M.J.W., T.A.G., J.F.D., R.K.W., T.J.L.);
`and the Division of Biostatistics (J.B.) —
`all at Washington University, St. Louis. Ad-
`dress reprint requests to Dr. Ley at Wash-
`ington University, 660 S. Euclid Ave.,
`Campus Box 8007, St. Louis, MO 63110,
`or at timley@wustl.edu.
`
`This article (10.1056/NEJMoa0903840) was
`published on August 5, 2009, at NEJM.org.
`
`N Engl J Med 2009;361:1058-66.
`Copyright © 2009 Massachusetts Medical Society.
`
`1058
`
`Background
`The full complement of DNA mutations that are responsible for the pathogenesis of
`acute myeloid leukemia (AML) is not yet known.
`Methods
`We used massively parallel DNA sequencing to obtain a very high level of coverage
`(approximately 98%) of a primary, cytogenetically normal, de novo genome for AML
`with minimal maturation (AML-M1) and a matched normal skin genome.
`Results
`We identified 12 acquired (somatic) mutations within the coding sequences of genes
`and 52 somatic point mutations in conserved or regulatory portions of the genome.
`All mutations appeared to be heterozygous and present in nearly all cells in the
`tumor sample. Four of the 64 mutations occurred in at least 1 additional AML
`sample in 188 samples that were tested. Mutations in NRAS and NPM1 had been
`identified previously in patients with AML, but two other mutations had not been
`identified. One of these mutations, in the IDH1 gene, was present in 15 of 187 ad-
`ditional AML genomes tested and was strongly associated with normal cytogenetic
`status; it was present in 13 of 80 cytogenetically normal samples (16%). The other
`was a nongenic mutation in a genomic region with regulatory potential and conser-
`vation in higher mammals; we detected it in one additional AML tumor. The AML
`genome that we sequenced contains approximately 750 point mutations, of which
`only a small fraction are likely to be relevant to pathogenesis.
`Conclusions
`By comparing the sequences of tumor and skin genomes of a patient with AML-M1,
`we have identified recurring mutations that may be relevant for pathogenesis.
`n engl j med 361;11 nejm.org september 10, 2009
`
`The New England Journal of Medicine
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`

`Recurring Mutations Found by Sequencing an AML Genome
`
`A cute myeloid leukemia (AML) is a
`
`clonal hematopoietic disease caused by
`both inherited and acquired genetic alter-
`ations.1-3 Current AML classification and prog-
`nostic systems incorporate genetic information
`but are limited to known abnormalities that have
`previously been identified with the use of cytoge-
`netics, array comparative genomic hybridization
`(CGH), gene-expression profiling, and the rese-
`quencing of candidate genes (see the Glossary).
`The karyotyping of AML cells remains the
`most powerful predictor of the outcome in pa-
`tients with AML and is routinely used by clini-
`cians.4,5 As an adjunct to cytogenetic studies,
`small subcytogenetic amplifications and dele-
`tions can be identified with the use of genomic
`methods, such as single-nucleotide-polymorphism
`(SNP) array and array CGH platforms (see the
`Glossary). However, these techniques remain in-
`vestigational, and studies6-9 suggest that there
`are few recurrent acquired copy-number altera-
`tions in each AML genome. Gene-expression
`profiling has identified patients with known
`chromosomal lesions and genetic mutations and
`subgroups of patients with normal cytogenetic
`profiles who have variable clinical outcomes.10,11
`Expression profiling has yielded single-gene pre-
`dictors of outcome that are currently being
`evaluated for clinical use.12-16 Candidate-gene re-
`sequencing studies have also identified recurrent
`mutations in several genes — for example, genes
`encoding FMS-related tyrosine kinase 3 (FLT3)
`and nucleophosmin 1 (NPM1) — that can help to
`stratify patients with normal cytogenetic pro-
`files according to risk and to identify patients
`for targeted therapy (e.g., those with mutated
`FLT3).3,12,17 However, the revised classification
`systems are imperfect, suggesting that impor-
`tant genetic factors for the pathogenesis of AML
`remain to be discovered.
`We have previously described the sequence of
`an entire AML genome from a patient who had
`AML with minimal maturation (AML-M1) and a
`normal cytogenetic profile.18 Here we describe the
`genome sequence of another such tumor and re-
`curring mutations in additional AML tumors.
`
`Me thods
`
`Details regarding the methods for library produc-
`tion, DNA sequencing with the Illumina Genome
`Analyzer II,19 evaluation of sequence coverage,
`
`identification of sequence variants, validation of
`variants and determination of the prevalence of
`variants in the index AML tumor, and screening
`of additional AML samples are provided in the
`Supplementary Appendix, available with the full
`text of this article at NEJM.org. All the high-
`quality single-nucleotide variants (SNVs) that were
`found in tumor and skin samples from this pa-
`tient are available in the database of genotypes
`and phenotypes (dbGaP) of the National Center
`for Biotechnology Information (accession number,
`phs000159.v1.p1).
`
`R esults
`
`Case Report
`A previously healthy 38-year-old man of European
`ancestry presented with fatigue and a cough. The
`white-cell count was 39,800 cells per cubic milli-
`meter, with 97% blasts; the hemoglobin level was
`8.9 g per deciliter, and the platelet count was
`35,000 per cubic millimeter. A bone marrow ex-
`amination revealed 90% cellularity and 86% my-
`eloperoxidase-positive blasts (Fig. 1 in the Supple-
`mentary Appendix). Routine cytogenetic analysis
`of bone marrow samples revealed a normal 46,XY
`karyotype. There was no family history of leuke-
`mia. The patient’s mother had received the diag-
`nosis of breast cancer at the age of 60 years and
`of non-Hodgkin’s lymphoma at the age of 63
`years; her half-sister had received the diagnosis
`of breast cancer at the age of 50 years.
`Samples of the patient’s bone marrow and
`skin were banked for whole-genome sequencing
`under a protocol approved by the institutional re-
`view board at Washington University. The patient
`provided written informed consent.
`The patient was treated initially with a 7-day
`course of infusional cytarabine and with a 3-day
`course of daunorubicin. Within 5 weeks, he had
`complete morphologic remission and recovery of
`white-cell and platelet counts. The patient subse-
`quently received consolidation therapy with four
`cycles of high-dose cytarabine without any fur-
`ther antileukemic therapy. He remained in com-
`plete remission 3 years later.
`
`Characterization of the Tumor Genome
`DNA samples from the patient’s bone marrow
`sample at the time of initial presentation and a
`normal skin-biopsy specimen obtained after the
`patient’s disease was in remission were labeled
`
`n engl j med 361;11 nejm.org september 10, 2009
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`1059
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`T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`Glossary
`
`Build 36 of the human reference genome: The most current version of the assembled human genome reference sequence,
`available online at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
`
`Comparative genomic hybridization (CGH): A comparison of DNA abundance, throughout the genome, between two DNA
`samples to identify regions where DNA copies have been gained or lost.
`
`dbSNP: A publicly available database of known DNA variants, housed at the National Center for Biotechnology Informa-
`tion (www.ncbi.nlm.nih.gov/SNP).
`
`Diploid coverage: A metric used in whole-genome sequencing studies to describe the likelihood of detecting both alleles
`at any given nucleotide position in a genome.
`
`Genic: Regions of the genome that contain genes, including their exons and introns.
`
`Haploid coverage: A metric used in whole-genome sequencing studies to describe detection of each nucleotide posi-
`tion in a genome for at least one allele; “1× coverage” is equivalent to the size of the genome (e.g., approximately
`3 billion base pairs for the human genome).
`
`Next-generation sequencing: A variety of new techniques that have in common the generation of DNA sequence from
`single molecules of DNA, rather than pools of DNA templates; hundreds of millions of DNA fragments can be se-
`quenced at the same time on a single platform (massively parallel sequencing).
`
`Paired-end reads: DNA sequences that are produced on next-generation sequencing platforms by sequencing both ends
`of DNA fragments, resulting in higher confidence in assigning the sequence a position in the reference genome
`and allowing the detection of structural variations.
`
`Partial uniparental disomy: An acquired somatic recombination event that causes the duplication of a part of a chromo-
`some from one parent, resulting in a “copy-number neutral” loss of heterozygosity for a chromosomal segment.
`
`Resequencing: Obtaining the DNA sequence of additional members of a species for which a completed reference se-
`quence is known and to which comparisons can be made.
`
`Sequencing run: The sequence that is generated by a complete Illumina flow cell or a similar next-generation sequencing
`platform; one sequencing run generates many billions of base pairs of sequence.
`
`Single-nucleotide polymorphism (SNP): A position in the genome where some individuals in a population inherit a
`change in a single nucleotide that differs from the reference genome.
`
`Single-nucleotide variant (SNV): A difference in a DNA sequence of a sample at a single position in the genome, as
`compared with the reference genome; each variant may represent either an inherited or an acquired change.
`
`SNP array: A microarray-based assay system that allows for simultaneous measurement of nucleotide sequence and
`abundance in a DNA sample at possibly hundreds of thousands of positions in the genome.
`
`and genotyped with the use of the Affymetrix
`Genome-Wide Human SNP Array 6.0. The tumor
`genome had no detectable somatic copy-number
`alterations and no regions of partial uniparental
`disomy (Glossary, and Fig. 2 in the Supplemen-
`tary Appendix). RNA that was derived from the
`same bone marrow sample was analyzed with
`the use of the Affymetrix GeneChip Human Ge-
`nome U133 Plus 2.0 array, which revealed an ex-
`pression signature similar to that of many other
`cytogenetically normal marrow samples from pa-
`tients with AML-M1 (Fig. 2 in the Supplementary
`Appendix).
`
`Sequence Coverage and Potential Mutations
`We sequenced 69.9 billion base pairs (23.3× hap-
`loid coverage) from DNA libraries that we gener-
`ated from the tumor sample and 63.9 billion base
`pairs from libraries that we generated from the
`normal skin sample (21.3× haploid coverage)
`(Glossary and Table 1). Using Affymetrix 6.0 SNP
`
`arrays, we confirmed the detection of both alleles
`of 98.5% of the approximately 45,000 high-qual-
`ity heterozygous SNPs in the tumor sample and
`97.4% of the approximately 45,000 high-quality
`heterozygous SNPs in the skin sample.
`A summary of the sequence differences be-
`tween the patient’s tumor genome and National
`Center for Biotechnology Information build 36 of
`the human reference genome is shown in Figure 1
`(see the Glossary).20 We identified 3,872,936 SNVs
`in the tumor genome, of which 3,464,449 passed
`a stringent calling filter. Of these SNVs, 3,377,680
`(97.5%) were detected in the skin genome, indi-
`cating that they were inherited variants. Of the
`86,769 potentially novel somatic SNVs, 66,513 had
`been described previously.
`We binned the remaining 20,256 SNVs into
`four tiers, which are detailed in the Supplemen-
`tary Appendix. Briefly, tier 1 contains all changes
`in the amino acid coding regions of annotated
`exons, consensus splice-site regions, and RNA
`
`1060
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`n engl j med 361;11 nejm.org september 10, 2009
`
`The New England Journal of Medicine
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`Recurring Mutations Found by Sequencing an AML Genome
`
`genes (including microRNA genes). Tier 2 con-
`tains changes in highly conserved regions of the
`genome or regions that have regulatory poten-
`tial. Tier 3 contains mutations in the nonrepeti-
`tive part of the genome that does not meet tier 2
`criteria, and tier 4 contains mutations in the re-
`mainder of the genome. We tentatively identified
`113 potential tier 1 mutations, 749 potential tier 2
`mutations, 3188 potential tier 3 mutations, and
`16,206 potential tier 4 mutations. For each of the
`113 putative tier 1 variants, we amplified the
`genomic region containing the mutation from
`both tumor and skin, using a polymerase-chain-
`reaction (PCR) assay, and performed Sanger se-
`quencing. Of the 101 variants that were called
`with low confidence (the calling algorithm is
`summarized in the Supplementary Appendix),
`none were validated. Of the high-confidence vari-
`ants, 10 of 12 were validated as somatic muta-
`tions. Similarly, we tested 178 low-confidence
`calls for tier 2, and only one was validated. In
`contrast, 51 of 104 high-confidence tier 2 calls
`were validated. We did not carry out validation
`studies of variants in tiers 3 and 4.
`We also searched for somatic insertions and
`deletions (indels) using an algorithm described
`in the Supplementary Appendix. We identified
`142 potential somatic indels (28 deletions and 114
`insertions). Of these variants, 119 failed valida-
`tion (i.e., they were falsely positive) in Sanger se-
`quencing of the relevant PCR products, 21 were
`validated but were present in both tumor and
`skin, and 2 were validated as somatic mutations.
`One was a 4-bp insertion in exon 12 of the NPM1
`gene associated with aberrant cytoplasmic ex-
`pression of nucleophosmin (NPMc). This inser-
`tion creates a frameshift mutation and a truncated
`protein that is known to have altered cellular
`localization, as described previously.21 The second
`mutation was a 3-bp insertion in the gene en-
`coding centrosomal protein 170kDa (CEP170) at
`amino acid 177, predicted to result in the addi-
`tion of a leucine residue at this position.
`
`Tier 1 Mutations
`The genes with tier 1 mutations and the conse-
`quences of these mutations are summarized in
`Table 2, and in Table 1 in the Supplementary Ap-
`pendix. Both the NPMc insertion and the NRAS
`mutation have been described previously in AML
`genomes, and both are known to be relevant for
`pathogenesis.3 Mutations in IDH1 (encoding isoc-
`itrate dehydrogenase 1), which are predicted to
`
`Table 1. Sequence Coverage for Tumor and Skin Genomes.*
`
`Variable
`
`Sequencing runs — no.†
`
`Haploid coverage
`
`SNVs — no.
`
`Concordance with dbSNP build 129 —
`no. (%)
`
`High-quality heterozygous SNPs
`
`Tumor
`
`16.5
`
`23.3×
`
`Skin
`
`13.125
`
`21.3×
`
`3,464,465
`
`3,448,797
`
`3,053,215 (88.1)
`
`2,992,069 (86.8)
`
`By array — no.
`
`45,111
`
`44,778
`
`By sequence — no. (% of array)
`
`44,442 (98.5)
`
`43,629 (97.4)
`
`High-quality homozygous rare SNPs
`
`By array — no.
`
`28,295
`
`27,735
`
`By sequence — no. (% of array)
`
`28,252 (99.8)
`
`27,685 (99.8)
`
`* The term dbSNP denotes a National Center for Biotechnology Information
` database of known DNA variants, SNP single-nucleotide polymorphism, and
`SNV single-nucleotide variant.
`† A single sequencing run uses all eight lanes of an Illumina flow cell (see the
`Glossary).
`
`affect the arginine residue at position 132, are
`found in malignant gliomas but have not been
`reported in patients with AML and are rare in
`other tumor types.22-24 Variants of the nine other
`tier 1 genes are discussed in the Supplementary
`Appendix.
`Each of the 10 point mutations was amplified
`from tumor and skin samples by means of PCR,
`and the DNA species carrying the variant allele
`was assayed by sequencing the PCR products with
`the use of the Illumina platform. The entire ex-
`periment was replicated with amplified genomic
`DNA, with excellent concordance for all samples
`(Fig. 3 in the Supplementary Appendix). The vari-
`ant allele frequencies of the two insertions were
`determined by sequencing PCR products contain-
`ing these mutations. The representation of all
`but two of the mutations — in chromosome 19
`open reading frame 62 (C19orf62), an unannotated
`gene of unknown function, and CEP170 — was
`approximately 50%, suggesting that all the mu-
`tations were heterozygous and present in nearly
`all the cells in the tumor sample (Fig. 2A). Ten of
`the 12 genes in tier 1 had probe sets on the Affy-
`metrix U133 Plus 2.0 array, and 9 of 10 were de-
`tectably expressed (Table 1). We also assayed ex-
`pression of the 10 nonsynonymous mutant alleles
`by means of reverse-transcriptase PCR, using
`amplicons designed to span introns, followed by
`sequencing and counting of the sequenced PCR
`products. Eight of the mutant alleles were detected
`
`n engl j med 361;11 nejm.org september 10, 2009
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`T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`3,872,936 Tumor SNVs were detected
`
`3,464,449 SNVs passed Maq software
`SNP filter
`
`86,769 Had a somatic score above 15
`
`3,377,680 SNVs were also
`detected in skin
`
`66,513 Had been described
`previously
`
`20,256 Were novel potential somatic SNVs
`
`113 Tier 1 SNVs
`
`749 Tier 2 SNVs
`
`3188 Tier 3 SNVs
`
`16,206 Tier 4 SNVs
`
`101 Were low
`confidence
`
`12 Were high
`confidence
`
`178 Were low
`confidence
`
`104 Were high
`confidence
`
`467 Were low
`confidence but
`not tested
`
`441 Were high
`confidence
`
`901 Were high
`confidence
`
`0 Were validated
`somatic
`
`10 Were validated
`somatic
`
`1 Was validated
`somatic
`
`51 Were validated
`somatic
`
`Figure 1. Flow Chart for Identification of Somatic Point Mutations in the Acute Myeloid Leukemia Genome.
`Maq denotes Mapping and Assembly with Quality, SNP single-nucleotide polymorphism, and SNV single-nucleotide variant.
`
`at frequencies of 35 to 85%. However, for two of
`the mutations (in FREM2 and IMPG2) we did not
`detect complementary DNA carrying the variant
`allele (although we easily detected the wild-type
`allele), even though each variant was present in
`approximately 50% of the tumor DNA.
`The individual bases that were mutated were
`highly conserved for 10 of the 12 variants, and all
`but 1 were found in highly conserved regions of
`the genome. The Sorting Intolerant from Tolerant
`(SIFT) algorithm (which gauges the likely effect
`of genic mutations on protein function) predicted
`that the mutations in NRAS, IDH1, IMPG2, and
`ANKRD26 were deleterious.25 The splice-site muta-
`
`tion at the 3(cid:2) end of intron 4 of C19orf62 caused
`exon 5 to be skipped (data not shown).
`We then genotyped the tier 1 mutations in
`187 additional samples from patients with AML
`whose clinical characteristics have been described
`previously26 (Table 2 in the Supplementary Appen-
`dix). The NPMc mutation was previously shown
`to be present in 43 of 180 samples (23.9%), and
`activating NRAS mutations were present in 17 of
`182 samples (9.3%).26 We observed mutations in
`IDH1, which were predicted to cause substitution
`of the arginine residue at position 132, in 16 of
`188 samples: R132C in 8 samples, R132H in
`7 samples, and R132S in 1 sample (Table 2 in the
`
`1062
`
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`The New England Journal of Medicine
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`Recurring Mutations Found by Sequencing an AML Genome
`
`Table 2. Tier 1 Mutations.*
`
`Annotated
`Gene
`
`Mutation
`Type
`
`Annotation
`
`SIFT
`Prediction
`
`Conservation
`Score
`
`Base
`Conservation
`
`CDC42
`
`NRAS
`
`IDH1
`
`IMPG2
`
`Missense
`
`Missense
`
`Missense
`
`Missense
`
`S30L
`
`G12D
`
`R132C
`
`G834D
`
`Tolerated
`
`Deleterious
`
`Deleterious
`
`Deleterious
`
`ANKRD26
`
`Missense
`
`K1300N
`
`Deleterious
`
`Tolerated
`
`597
`
`616
`
`445
`
`472
`
`444
`
`539
`
`1
`
`1
`
`1
`
`0.018
`
`1
`
`0.946
`
`Variant Frequency
`
`Skin
`
`Tumor
`
`cDNA
`
`%
`
`49.27
`
`43.00
`
`46.06
`
`46.22
`
`51.73
`
`45.28
`
`1.03
`
`0.66
`
`0.81
`
`0.67
`
`0.70
`
`0.68
`
`46.3
`
`42.0
`
`63.9
`
`0.4
`
`33.1
`
`47.9
`
`Best
`Probe†
`
`27,990
`
`7,468
`
`11,400
`
`NA
`
`514
`
`12,138
`
`LTA4H
`
`FREM2
`
`C19orf62
`
`SRRM1
`
`PCDHA6
`
`CEP170
`
`NPM1
`
`Missense
`
`Missense
`
`Splice-site
`
`Silent
`
`Silent
`
`In-frame
`insertion
`
`Frame-shift
`insertion
`
`F107S
`
`Q2077E
`
`Exon 5-1
`
`P691
`
`A731
`
`Codon 177
`in-frame ins L
`
`W288fs
`
`Tolerated
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`464
`
`444
`
`553
`
`NS
`
`513
`
`689
`
`1
`
`1
`
`0.988
`
`0.423
`
`1
`
`1
`
`0.37
`
`0.27
`
`0.97
`
`0.66
`
`0.28
`
`48.92
`
`38.71
`
`46.61
`
`49.75
`
`28.57
`
`0‡
`
`38.8
`
`ND
`
`ND
`
`52.0
`
`NA
`
`5,021
`
`12,858
`
`Absent
`
`15,298
`
`0
`
`45.46
`
`85.4
`
`27,150
`
`* The term cDNA denotes complementary DNA, ins L insertion of Leu, NA not available, ND not done, NS no score, and SIFT Sorting
`Intolerant from Tolerant.
`† The best probe refers to the signal value for the most highly expressed probe on the Affymetrix GeneChip Human Genome U133 Plus 2.0
`array, transformed by statistical algorithms (MAS 5.0).
`‡ The variant frequency was calculated from cDNA subclones.
`
`Supplementary Appendix). The other nine muta-
`tions were not detected in the 187 additional
`samples. We detected no R172 mutations in IDH2
`in 188 samples (the sample from the index pa-
`tient and the 187 additional samples), nor did we
`observe additional mutations in any of the exons
`of IDH1 or CDC42.
`A nonsynonymous acquired mutation (C328Y)
`was found in the mitochondrial gene ND4, which
`encodes NADH dehydrogenase subunit 4, a part
`of complex 1 of the electron transport chain.
`Two of 93 additional AML samples also had non-
`synonymous mutations in this gene, but the im-
`portance of these mutations is not yet clear
`(Table 5 and the Results and Discussion section
`in the Supplementary Appendix).
`
`Tier 2 Mutations
`We confirmed 52 mutations in tier 2. DNA seg-
`ments, each containing 1 of the 52 mutations, were
`PCR-amplified from the tumor and skin samples
`and sequenced to determine the proportion of DNA
`molecules carrying the mutation (Fig. 2B, and
`Table 4 in the Supplementary Appendix). Three of
`
`these tier 2 mutations had variant frequencies of
`approximately 98%, and all were located on chro-
`mosome X or Y. Because only a single copy of
`these chromosomes was present in this male ge-
`nome, the high representation of these three tier 2
`mutations was consistent with the finding that
`an extremely high percentage of cells within the
`bone marrow sample were part of the malignant
`clone. One mutation (chromosome 4 at position
`128,102,994) had a variant read frequency of ap-
`proximately 78%, and we observed no somatic
`microamplification or deletion near this variant.
`Of the tier 2 mutations, 39 were present in approx-
`imately 50% of DNA species, and 9 were present
`in approximately 40%. We genotyped the 52 tier 2
`mutations in 187 additional AML samples and de-
`tected the presence of just 1 of the mutations (on
`chromosome 10) in 1 other AML sample, from a
`patient with myelomonocytic leukemia (AML-M4),
`which bore a translocation and did not have a
`paired normal sample (Table 2 in the Supplemen-
`tary Appendix). The proportion of DNA species in
`this sample that carried the mutation was 54%,
`suggesting that it was heterozygous.
`
`n engl j med 361;11 nejm.org september 10, 2009
`
`1063
`
`The New England Journal of Medicine
`Downloaded from nejm.org on August 2, 2022. For personal use only. No other uses without permission.
` Copyright © 2009 Massachusetts Medical Society. All rights reserved.
`
`Rigel Exhibit 1007
`Page 6 of 9
`
`

`

`T h e ne w e ngl a nd jou r na l o f m e dic i ne
`
`detected only in patients with cytogenetic profiles
`associated with intermediate risk (P<0.001).4,5
`Although the patients who were analyzed in this
`study were not treated with a single uniform pro-
`tocol, outcome data were available for all 188 pa-
`tients (Table 2 in the Supplementary Appendix).
`IDH1 mutational status did not have independent
`prognostic value with respect to overall survival
`in multivariate analysis; subgroup analysis showed
`a possible adverse effect on overall survival among
`patients with normal-karyotype AML and wild-
`type NPM1, regardless of FLT3 status (Fig. 4 in
`the Supplementary Appendix).
`
`Discussion
`
`Our findings support the use of an unbiased se-
`quencing approach to discover previously unsus-
`pected, recurring mutations in a cancer genome.
`With improved sequencing techniques, we covered
`this genome more completely than the first one
`we sequenced (98% vs. 91% diploid coverage) and
`used fewer sequencing runs (16.5 vs. 98), resulting
`in a dramatically reduced cost of data generation.
`With better data quality and calling algorithms,
`we reduced the 96% false positive frequency of
`possible mutations for the first sequenced AML
`genome to a frequency of 47% of the high-confi-
`dence tier 1 and 2 mutations called in this genome.
`We predicted 1458 tumor-specific point mutations
`with high confidence; we tested 116 of these with
`validation sequencing and confirmed 61 of them
`(53%). Thus, this genome may contain approxi-
`mately 750 somatic point mutations. We detected
`mutations in NRAS, NPMc, and IDH1 and a tier 2
`mutation on chromosome 10 in more than one
`AML genome, suggesting that these mutations
`are not random and are probably important for
`the pathogenesis of this tumor.
`We suggest that the 12 nonsynonymous mu-
`tations are the most likely to be relevant for
`pathogenesis, since they could potentially alter
`the function of expressed genes. Consistent with
`this idea and with the results of our previous
`study18 is the finding that all these mutations
`were retained in the dominant clone. Surprisingly,
`we found that virtually all the 52 tier 2 muta-
`tions were also present in nearly every tumor cell
`in the sample, suggesting that they are also a
`part of the same dominant clone. However, one
`cannot conclude that these mutations (or any of
`the tier 3 or 4 mutations) are relevant for patho-
`
`(cid:26) (cid:39)(cid:8)(cid:55)(cid:56)(cid:55)(cid:8)(cid:10)(cid:6)(cid:55)(cid:7)(cid:11)(cid:55)(cid:50)(cid:11)(cid:4)(cid:5)(cid:8)(cid:6)(cid:5)(cid:7)(cid:10)(cid:9)
`Tumor DNA
`
`Tumor cDNA
`
`Skin DNA
`
`100
`
`80
`
`60
`
`40
`
`20
`
`(cid:4)(cid:5)(cid:8)(cid:6)(cid:5)(cid:7)(cid:10)(cid:9)(cid:11)(cid:2)(cid:1)(cid:3)
`
`0
`
`N P M 1c
`CEP170 177insL
`N RAS (cid:32)(cid:16)(cid:17)(cid:29)
`CD C42 (cid:42)(cid:18)(cid:15)(cid:36)
`LTA4H (cid:31)(cid:16)(cid:15)(cid:22)(cid:42)
`IM PG2 (cid:32)(cid:23)(cid:18)(cid:19)(cid:29)
`FRE M 2 (cid:40)(cid:17)(cid:15)(cid:22)(cid:22)(cid:30)
`(cid:8)(cid:9)(cid:24)(cid:21)(cid:18)(cid:21)(cid:16)(cid:19)(cid:21)
`
`(cid:8)(cid:9)(cid:21)(cid:15)(cid:22)(cid:15)(cid:17)(cid:16)(cid:23)(cid:18)
`(cid:8)(cid:9)(cid:16)(cid:17)(cid:18)(cid:20)(cid:23)(cid:23)(cid:23)(cid:22)
`A N KRD26 (cid:35)(cid:16)(cid:18)(cid:15)(cid:15)(cid:37)
`ID H1 (cid:41)(cid:16)(cid:18)(cid:17)(cid:28)
`C19orf62 (cid:49)(cid:20)(cid:13)(cid:16)
`
`
`
`(cid:8)(cid:9)(cid:22)(cid:18)(cid:24)(cid:21)(cid:18)(cid:24)(cid:22)(cid:8)(cid:9)(cid:21)(cid:24)(cid:21)(cid:21)(cid:16)(cid:20)(cid:15)(cid:8)(cid:9)(cid:17)(cid:24)(cid:21)(cid:15)(cid:21)(cid:19)(cid:17)
`
`(cid:27) (cid:4)(cid:5)(cid:8)(cid:6)(cid:5)(cid:7)(cid:10)(cid:11)(cid:31)(cid:8)(cid:49)(cid:57)(cid:58)(cid:49)(cid:7)(cid:47)(cid:60)
`100
`
`Tumor
`Skin
`
`80
`
`60
`
`40
`
`20
`
`0
`
`(cid:4)(cid:5)(cid:8)(cid:6)(cid:5)(cid:7)(cid:10)(cid:9)(cid:11)(cid:2)(cid:1)(cid:3)
`
`(cid:43)(cid:6)(cid:49)(cid:8)(cid:11)(cid:16)
`
`(cid:43)(cid:6)(cid:49)(cid:8)(cid:11)(cid:17)
`
`(cid:28)(cid:55)(cid:7)(cid:10)(cid:8)(cid:55)(cid:54)(cid:11)(cid:42)(cid:37)(cid:39)(cid:9)
`
`Figure 2. Allele Frequency in Tumor DNA, Tumor Complementary DNA,
`and Skin DNA.
`Panel A shows the percentage of variant alleles that were detected in tumor
`DNA, tumor complementary DNA (cDNA), and skin DNA for the 10 validated
`nonsynonymous tier 1 somatic mutations in the index patient. For compari-
`son, variant allele frequencies are shown for six known single-nucleotide poly-
`morphisms (SNPs). The patient was homozygous for the reference sequence
`for the first two variants, heterozygous for the next two variants, and homozy-
`gous for a rare SNP for the last two variants. Panel B shows variant allele
`frequencies for all validated tier 1 and tier 2 mutations and the six control
`SNPs for tumor DNA and skin DNA.
`
`Patients with the IDH1 Mutation
`Of the 16 patients who had AML with an IDH1
`R132 mutation, 13 had tumors with normal cyto-
`genetic profiles (of a total of 80 cytogenetically
`normal samples [16%]), 2 had trisomy 8, and
`1 had trisomy 13. Ten of the 16 patients had AML-
`M1, three had AML with maturation (AML-M2),
`and three had AML-M4. The characteristics of
`patients with and those without the IDH1 muta-
`tion are shown in Table 3, and in Tables 2 and 3 in
`the Supplementary Appendix. The mutation was
`
`1064
`
`n engl j med 361;11 nejm.org september 10, 2009
`
`The New England Journal of Medicine
`Downloaded from nejm.org on August 2, 2022. For personal use only. No other uses without permission.
` Copyright © 2009 Massachusetts Medical Society. All rights reserved.
`
`Rigel Exhibit 1007
`Page 7 of 9
`
`

`

`Recurring Mutations Found by Sequencing an AML Genome
`
`genesis simply because they are found at a high
`frequency in the dominant clone. It is more likely
`that most of these mutations are random, benign
`sequence changes that existed in the hematopoi-
`etic cell that was transformed (i.e., they were
`preexisting and carried along as benign “passen-
`gers,” irrelevant for pathogenesis). The finding
`that the percentage of mutations found in each
`tier closely approximated the total amount of
`DNA assayed in that tier supports this hypothe-
`sis. Collectively, these data suggest that the vast
`majority of the mutations that we detected in this
`genome are random, background mutations in the
`hematopoietic stem cell that was transformed.27
`Functional validation will be required to prove
`which mutations are truly important.
`The best test of the relevance of individual
`mutations for pathogenesis (in the absence of
`functional validation