Lay SummaryAcute myeloid leukemia is a highly malignant hematopoietic tumor that affects about 13,000 adults yearly in the United States. The treatment of this disease has changed little in the past two decades, since most of the genetic events that initiate the disease remain undiscovered. Whole genome sequencing is now possible at a reasonable cost and timeframe to utilize this approach for unbiased discovery of tumor-specific somatic mutations that alter the protein-coding genes. Here we show the results obtained by sequencing a typical acute myeloid leukemia genome and its matched normal counterpart, obtained from the patient’s skin. We discovered 10 genes with acquired mutations; two were previously described mutations thought to contribute to tumor progression, and 8 were novel mutations present in virtually all tumor cells at presentation and relapse, whose function is not yet known. Our study establishes whole genome sequencing as an unbiased method for discovering initiating mutations in cancer genomes, and for identifying novel genes that may respond to targeted therapies.We used massively parallel sequencing technology to sequence the genomic DNA of tumor and normal skin cells obtained from a patient with a typical presentation of FAB M1 Acute Myeloid Leukemia (AML) with normal cytogenetics. 32.7-fold ‘haploid’ coverage (98 billion bases) was obtained for the tumor genome, and 13.9-fold coverage (41.8 billion bases) was obtained for the normal sample. Of 2,647,695 well-supported Single Nucleotide Variants (SNVs) found in the tumor genome, 2,588,486 (97.7%) also were detected in the patient’s skin genome, limiting the number of variants that required further study. For the purposes of this initial study, we restricted our downstream analysis to the coding sequences of annotated genes: we found only eight heterozygous, non-synonymous somatic SNVs in the entire genome. All were novel, including mutations in protocadherin/cadherin family members (CDH24 and PCLKC), G-protein coupled receptors (GPR123 and EBI2), a protein phosphatase (PTPRT), a potential guanine nucleotide exchange factor (KNDC1), a peptide/drug transporter (SLC15A1), and a glutamate receptor gene (GRINL1B). We also detected previously described, recurrent somatic insertions in the FLT3 and NPM1 genes. Based on deep readcount data, we determined that all of these mutations (except FLT3) were present in nearly all tumor cells at presentation, and again at relapse 11 months later, suggesting that the patient had a single dominant clone containing all of the mutations. These results demonstrate the power of whole genome sequencing to discover novel cancer-associated mutations.

Activating mutations in tyrosine kinase (TK) genes (eg, FLT3 and KIT) are found in more than 30% of patients with de novo acute myeloid leukemia (AML); many groups have speculated that mutations in other TK genes may be present in the remaining 70%. We performed highthroughput resequencing of the kinase domains of 26 TK genes (11 receptor TK; 15 cytoplasmic TK) expressed in most AML patients using genomic DNA from the bone marrow (tumor) and matched skin biopsy samples ("germline") from 94 patients with de novo AML; sequence variants were validated in an additional 94 AML tumor samples (14.3 million base pairs of sequence were obtained and analyzed). We identified known somatic mutations in FLT3, KIT, and JAK2 TK genes at the expected frequencies and found 4 novel somatic mutations, JAK1 V623A , JAK1 T478S , DDR1 A803V , and NTRK1 S677N , once each in 4 respective patients of 188 tested. We also identified novel germline sequence changes encoding amino acid substitutions (ie, nonsynonymous changes) in 14 TK genes, including TYK2, which had the largest number of nonsynonymous sequence variants (11 total detected). Additional studies will be required to define the roles that these somatic and germline TK gene variants play in AML pathogenesis. (Blood. 2008; 111:4797-4808)

Cytogenetic analysis of acute myeloid leukemia (AML) cells has accelerated the identification of genes important for AML pathogenesis. To complement cytogenetic studies and to identify genes altered in AML genomes, we performed genome-wide copy number analysis with paired normal and tumor DNA obtained from 86 adult patients with de novo AML using 1.85 million feature SNP arrays. Acquired copy number alterations (CNAs) were confirmed using an ultra-dense array comparative genomic hybridization platform. A total of 201 somatic CNAs were found in the 86 AML genomes (mean, 2.34 CNAs per genome), with French-AmericanBritish system M6 and M7 genomes containing the most changes (10 -29 CNAs per genome). Twenty-four percent of AML patients with normal cytogenetics had CNA, whereas 40% of patients with an abnormal karyotype had additional CNA detected by SNP array, and several CNA regions were recurrent. The mRNA expression levels of 57 genes were significantly altered in 27 of 50 recurrent CNA regions <5 megabases in size. A total of 8 uniparental disomy (UPD) segments were identified in the 86 genomes; 6 of 8 UPD calls occurred in samples with a normal karyotype. Collectively, 34 of 86 AML genomes (40%) contained alterations not found with cytogenetics, and 98% of these regions contained genes. Of 86 genomes, 43 (50%) had no CNA or UPD at this level of resolution. In this study of 86 adult AML genomes, the use of an unbiased high-resolution genomic screen identified many genes not previously implicated in AML that may be relevant for pathogenesis, along with many known oncogenes and tumor suppressor genes. AML ͉ array CGH ͉ genomics ͉ SNP arrayA cute myeloid leukemia (AML) is a heterogeneous group of diseases currently classified by abnormalities in bone marrow morphology, karyotype, acquired gene mutations, and alterations in gene expression (1-3). Although the identification of specific gene mutations has resulted in improved treatments and outcomes for some AML patients (4), enormous clinical heterogeneity exists and may reflect the presence of as-yet undetected initiating and cooperating mutations. Therefore, the discovery of somatic mutations in the genomes of AML patients with normal and abnormal karyotypes will advance our understanding of the genetics underlying AML and should lead to more specific therapies and better patient classification schemes.The discovery of previously uncharacterized genes mutated in acute lymphoblastic leukemia (ALL) was recently reported using SNP array technology for DNA copy number analysis (5). SNP array platforms can detect genomic amplifications, deletions, SNP loss of heterozygosity (LOH), and regions of uniparental disomy (UPD) (copy-neutral LOH events) in cancer cells. Early studies using SNP arrays and array comparative genomic hybridization (CGH) platforms have suggested that both copy number alterations (CNAs) and UPD are common in AML genomes (6-12). However, these studies used low-resolution arrays, often used reference DNA that was not obtained from the same patient's n...

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis. Like most other bone marrow failure syndromes, it is associated with a marked propensity to transform into a myelodysplastic syndrome (MDS) or acute leukemia, with a cumulative rate of transformation to MDS/leukemia that exceeds 20%. The genetic (and/or epigenetic) changes that contribute to malignant transformation in SCN are largely unknown. In this study, we performed mutational profiling of 14 genes previously implicated in leukemogenesis using 14 MDS/leukemia samples from patients with SCN. We used high-throughput exon-based resequencing of whole-genome-amplified genomic DNA with a semiautomated method to detect mutations. The sensitivity and specificity of the sequencing pipeline was validated by determining the frequency of mutations in these 14 genes using 188 de novo AML samples. As expected, mutations of tyrosine kinase genes (FLT3, KIT, and JAK2) were common in de novo AML, with a cumulative frequency of 30%. In contrast, no mutations in these genes were detected in the SCN samples; instead, mutations of CSF3R, encoding the G-CSF receptor, were common. These data support the hypothesis that mutations of CSF3R may provide the "activated tyrosine kinase signal" that is thought to be important for leukemogenesis. (Blood. 2007;110: [1648][1649][1650][1651][1652][1653][1654][1655]

Somatic mutations in JAK2 are frequently found in myeloproliferative diseases, and gain-of-function JAK3 alleles have been identified in M7 acute myeloid leukemia (AML), but a role for JAK1 in AML has not been described. We screened the entire coding region of JAK1 by total exonic resequencing of bone marrow DNA samples from 94 patients with de novo AML. We identified 2 novel somatic mutations in highly conserved residues of the JAK1 gene (T478S, V623A), in 2 separate patients and confirmed these by resequencing germ line DNA samples from the same patients. Overexpression of mutant JAK1 did not transform primary murine cells in standard assays, but compared with wild-type JAK1, JAK1 T478S , and JAK1 V623A expression was associated with increased STAT1 activation in response to type I interferon and activation of multiple downstream signaling pathways. This is the first report to demonstrate somatic JAK1 mutations in AML and suggests that JAK1 mutations may function as disease-modifying mutations in AML pathogenesis. IntroductionThe Janus kinase (JAK) genes encode nonreceptor tyrosine kinases, including 4 family members JAK1, JAK2, JAK3, and TYK2. 1 Ligand binding to cytokine receptor results in transphosphorylation of JAK kinases, and activated JAK kinases in turn phosphorylate receptor intracellular domain tyrosines to create docking sites to recruit SH2-domain-containing downstream signaling molecules-especially the signal transducers and activators of transcription (STATs). 2 The first genetic evidence implicating constitutive JAK-STAT activation in oncogenesis was derived from studies of signal transduction in the fruit fly. Fruit flies expressing constitutively activated Drosophila melanogaster JAK homolog, hopscotch, develop a hematopoietic neoplasia resembling leukemia. 3,4 Dysregulation of the JAK-STAT signaling pathway has been described in a variety of malignancies, including hematopoietic neoplasms. 5 Direct evidence for aberrant JAK activation in human tumorigenesis was first confirmed by the cloning of the t(9;12) translocation breakpoint in acute lymphocytic leukemia and the identification of the TEL-JAK2 fusion protein. 6 The somatic JAK2 mutation V617F has been reported in most patients with polycythemia vera (PV) as well as in approximately one third of patients with essential thrombocythemia and or idiopathic myelofibrosis. 7-10 The JAK2 V617F protein demonstrates constitutively activated kinase activity in vitro and coexpression of JAK2 V617F with type I cytokine receptors in Ba/F3 cells resulted in cytokine-independent JAK-STAT signaling and growth factor-independent cell growth. 11Additional JAK2 gain-of-function mutations have been identified in PV, idiopathic erythrocytosis, and acute leukemia. 12,13 Furthermore, activating alleles of JAK3 have been reported in association with acute megakaryoblastic leukemia. 14 JAK1 plays important roles in cytokine signal transduction 15 but has not been implicated in leukemia development. We sought to evaluate the frequency and the possible contributi...