Clonal architecture of secondary acute myeloid leukemia

BACKGROUND: The myelodysplastic syndromes are a group of hematologic disorders that often evolve into secondary acute myeloid leukemia (AML). The genetic changes that underlie progression from the myelodysplastic syndromes to secondary AML are not well understood. METHODS: We performed whole-genome sequencing of seven paired samples of skin and bone marrow in seven subjects with secondary AML to identify somatic mutations specific to secondary AML. We then genotyped a bone marrow sample obtained during the antecedent myelodysplastic-syndrome stage from each subject to determine the presence or absence of the specific somatic mutations. We identified recurrent mutations in coding genes and defined the clonal architecture of each pair of samples from the myelodysplastic-syndrome stage and the secondary-AML stage, using the allele burden of hundreds of mutations. RESULTS: Approximately 85% of bone marrow cells were clonal in the myelodysplastic-syndrome and secondary-AML samples, regardless of the myeloblast count. The secondary-AML samples contained mutations in 11 recurrently mutated genes, including 4 genes that have not been previously implicated in the myelodysplastic syndromes or AML. In every case, progression to acute leukemia was defined by the persistence of an antecedent founding clone containing 182 to 660 somatic mutations and the outgrowth or emergence of at least one subclone, harboring dozens to hundreds of new mutations. All founding clones and subclones contained at least one mutation in a coding gene. CONCLUSIONS: Nearly all the bone marrow cells in patients with myelodysplastic syndromes and secondary AML are clonally derived. Genetic evolution of secondary AML is a dynamic process shaped by multiple cycles of mutation acquisition and clonal selection. Recurrent gene mutations are found in both founding clones and daughter subclones. (Funded by the National Institutes of Health and others.

Integrated genomic characterization of endometrial carcinoma

SummaryWe performed an integrated genomic, transcriptomic, and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumors and ~25% of high-grade endometrioid tumors have extensive copy number alterations, few DNA methylation changes, low ER/PR levels, and frequent TP53 mutations. Most endometrioid tumors have few copy number alterations or TP53 mutations but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A, KRAS and novel mutations in the SWI/SNF gene ARID5B. A subset of endometrioid tumors we identified had a dramatically increased transversion mutation frequency, and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy number low, and copy number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may impact post-surgical adjuvant treatment for women with aggressive tumors

Comprehensive molecular portraits of human breast tumours

This Article from the Cancer Genome Atlas consortium describes a multifaceted analysis of primary breast cancers in 825 people. Exome sequencing, copy number variation, DNA methylation, messenger RNA arrays, microRNA sequencing and proteomic analyses were performed and integrated to shed light on breast-cancer heterogeneity. Just three genes — TP53, PIK3CA and GATA3 — are mutated at greater than 10% frequency across all breast cancers. Many subtype-associated and novel mutations were identified, as well as two breast-cancer subgroups with specific signalling-pathway signatures. The analyses also suggest that much of the clinically observable plasticity and heterogeneity occurs within, and not across, the major subtypes of breast cancer

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Multiplatform Analysis of 12 Cancer Types Reveals Molecular Classification within and across Tissues of Origin

Recent genomic analyses of pathologically-defined tumor types identify “within-a-tissue” disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head & neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multi-platform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All datasets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies

Recurrent DNMT3A Mutations In Patients with Myelodysplastic Syndrome

Abstract Abstract 608 Myelodysplastic syndrome (MDS) genomes are characterized by global DNA hypomethylation with concomitant hypermethylation of gene promoter regions compared to CD34+ cells from normal bone marrow samples. Currently, the underlying mechanism of altered DNA methylation in MDS genomes and the critical target genes affected by methylation remain largely unknown. The methylation of CpG dinucleotides in humans is mediated by DNA methyltransferases, including DNMT1, DNMT3A, and DNMT3B. DNMT3A and DNMT3B are the dominant DNA methyltransferases involved in de novo DNA methylation and act independent of replication, whereas DNMT1 acts predominantly during replication to maintain hemimethylated DNA. The function of these proteins in cancer cells is less well defined. Our group recently found that DNMT3A mutations are common in de novo acute myeloid leukemia (62/281 cases, 22%) and are associated with poor survival (Ley, et al, unpublished), providing a rationale for examining the mutation status of DNMT3A in MDS patients. MDS cases (n=150) were classified according to the French-American-British (FAB) system. The patients included refractory anemia (RA; n=67), RA with ringed sideroblasts (RARS; n=5), RA with excess blasts (RAEB; n=72), and RA with excess blasts in transformation (RAEB-T; n=6). The median International Prognostic Scoring System (IPSS) score was 1 (range 0–3), and the median myeloblast count was 4 (range 0–28%). We designed and validated 28 primer pairs covering the coding sequences and splice sites of all 23 exons for DNMT3A. Paired DNA samples were obtained from the bone marrow (tumor) and skin (normal) of each patient so that somatic mutations could be distinguished from inherited variants/polymorphisms. 17,120 reads were produced by capillary sequencing, providing at least 1X coverage for 82.6% of the target sequence (low/no coverage was obtained for 2 out of 28 amplicons). A semiautomated analysis pipeline was used to identify sequence variants and we restricted our analysis to nonsynonymous and splice site nucleotide changes. All mutations were confirmed by independent PCR and sequencing. We identified nonsynonymous DNMT3A mutations in 12/150 bone marrow samples (8% of cases). All the mutations were heterozygous (10 missense, 1 nonsense, 1 frameshift) and were computationally predicted (by SIFT and/or PolyPhen2) to have deleterious functional consequences. DNMT3A mRNA is expressed in normal CD34+ bone marrow cells and was expressed in all MDS patient samples tested (n=28), independent of mutation status. There was no difference in the expression level of total DNMT3A mRNA in CD34+ cells harvested from mutant (n=3) vs. non-mutant MDS samples (n=25). Amino acid R882, located in the methyltransferase domain of DNMT3A, was the most common mutation site, accounting for 4/12 mutations. The clinical characteristics of the 12 patients with DNMT3A mutations were similar to those of the 138 patients without mutations. Specifically, DNMT3A mutations were present in all MDS FAB subtypes (excluding CMML which was not tested) and in patients with IPSS scores ranging from 0–3. Mutations were not associated with a specific karyotype. In addition, there was no correlation between mutation detection and the myeloblast count of the banked bone marrow specimen, suggesting that mutations were not missed due to the cellular heterogeneity in the samples. We compared the overall (OS) and event-free survival (EFS) of the 12 patients with DNMT3A mutations vs. 138 patients without a mutation and observed a significantly worse OS in patients with mutations (p=0.02), with a median survival of 433 and 945 days, respectively. There was a trend towards worse EFS for patients with mutations (p=0.05). A multivariate analysis for outcomes could not be performed due to the small sample size of patients with mutations, indicating that a larger cohort from a clinical trial will be needed to properly address the affect of DNMT3A mutations on outcomes. The small sample size also precluded us from addressing whether the response to the hypomethylating agents 5-azacytidine or decitabine correlated with the mutation status of DNMT3A. If validated in larger cohort studies, we propose that DNMT3A mutation status could help risk stratify de novo MDS patients for more aggressive treatment early in their disease course. Disclosures: Westervelt: Novartis: Honoraria; Celgene: Honoraria, Speakers Bureau. DiPersio:Genzyme: Honoraria. </jats:sec

Background Mutations in Parental Cells Account for Most of the Genetic Heterogeneity of Induced Pluripotent Stem Cells

SummaryTo assess the genetic consequences of induced pluripotent stem cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. These data suggest that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which “captures” their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method

Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein(1,2). Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis

Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing

Most patients with acute myeloid leukemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level(1,2). To determine the mutational spectrum associated with relapse, we sequenced the primary tumor and relapse genomes from 8 AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to precisely define clonality and clonal evolution patterns at relapse. Besides discovering novel, recurrently mutated genes (e.g. WAC, SMC3, DIS3, DDX41, and DAXX) in AML, we found two major clonal evolution patterns during AML relapse: 1) the founding clone in the primary tumor 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 vs. primary tumor mutations in all 8 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