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Cytogenetics and gene mutations influence survival in older patients with acute myeloid leukemia treated with azacitidine or conventional care.
Older patients with newly diagnosed acute myeloid leukemia (AML) in the phase 3 AZA-AML-001 study were evaluated at entry for cytogenetic abnormalities, and a subgroup of patients was assessed for gene mutations. Patients received azacitidine 75 mg/m2/day x7 days (n = 240) or conventional care regimens (CCR; n = 245): intensive chemotherapy, low-dose cytarabine, or best supportive care only. Overall survival (OS) was assessed for patients with common (occurring in ≥10% of patients) cytogenetic abnormalities and karyotypes, and for patients with recurring gene mutations. There was a significant OS improvement with azacitidine vs CCR for patients with European LeukemiaNet-defined Adverse karyotype (HR 0.71 [95%CI 0.51-0.99]; P = 0.046). Azacitidine-treated patients with -5/5q-, -7/7q-, or 17p abnormalities, or with monosomal or complex karyotypes, had a 31-46% reduced risk of death vs CCR. The most frequent gene mutations were DNMT3A (27%), TET2 (25%), IDH2 (23% [R140, 15%; R172, 8%]), and TP53 (21%). Compared with wild-type, OS was significantly reduced among CCR-treated patients with TP53 or NRAS mutations and azacitidine-treated patients with FLT3 or TET2 mutations. Azacitidine may be a preferred treatment for older patients with AML with Adverse-risk cytogenetics, particularly those with chromosome 5, 7, and/or 17 abnormalities and complex or monosomal karyotypes. The influence of gene mutations in azacitidine-treated patients warrants further study.
Pan-cancer analysis of whole genomes.
Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale1-3. Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter4; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation5,6; analyses timings and patterns of tumour evolution7; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity8,9; and evaluates a range of more-specialized features of cancer genomes8,10-18.
GATA1 and cooperating mutations in myeloid leukaemia of Down syndrome.
Myeloid leukaemia of Down syndrome (ML-DS) is an acute megakaryoblastic/erythroid leukaemia uniquely found in children with Down syndrome (constitutive trisomy 21). It has a unique clinical course, being preceded by a pre-leukaemic condition known as transient abnormal myelopoiesis (TAM), and provides an excellent model to study multistep leukaemogenesis. Both TAM and ML-DS blasts carry acquired N-terminal truncating mutations in the erythro-megakaryocytic transcription factor GATA1. These result in exclusive production of a shorter isoform (GATA1s). The majority of TAM cases resolve spontaneously without the need for treatment; however, around 10% acquire additional cooperating mutations and transform to leukaemia, with differentiation block and clinically significant cytopenias. Transformation is driven by the acquisition of additional mutation(s), which cooperate with GATA1s to perturb normal haematopoiesis.
The interaction of alpha thalassaemia and sickle cell-beta zero thalassaemia.
The effects of alpha thalassaemia on sickle cell-beta zero thalassaemia have been studied by comparing haematological and clinical features in four subjects homozygous for alpha thalassaemia 2 (2-gene group), 27 heterozygotes (3-gene group), and 55 with a normal alpha globin gene complement (4-gene group). Alpha thalassaemia was associated with significantly higher haemoglobin levels and lower reticulocyte counts independent of the presence of splenomegaly. Contrary to expectation, alpha thalassaemia was associated with small but significant increases in mean cell volume and mean corpuscular haemoglobin concentration. Splenomegaly at age 5 years and episodes of acute splenic sequestration were significantly more frequent in the 4-gene group. There were no significant differences in painful crises, acute chest syndrome, or other clinical features.
Cis-acting sequences regulating expression of the human alpha-globin cluster lie within constitutively open chromatin.
Current models suggest that tissue-specific genes are arranged in discrete, independently controlled segments of chromatin referred to as regulatory domains. Transition from a closed to open chromatin structure may be an important step in the regulation of gene expression. To determine whether the human alpha-globin cluster, like the beta-globin cluster, lies within a discrete, erythroid-specific domain, we have examined the long-range genomic organization and chromatin structure around this region. The alpha genes lie adjacent to at least four widely expressed genes. The major alpha-globin regulatory element lies 40 kb away from the cluster within an intron of one of these genes. Therefore, unlike the beta cluster, cis-acting sequences controlling alpha gene expression are dispersed within a region of chromatin that is open in both erythroid and nonerythroid cells. This implies a difference in the hierarchical control of alpha- and beta-globin expression.
Contrasting effects of alpha and beta globin regulatory elements on chromatin structure may be related to their different chromosomal environments.
Expression of the human alpha and beta globin gene clusters is regulated by remote sequences, referred to as HS -40 and the beta-locus control region (beta-LCR) that lie 5-40 kb upstream of the genes they activate. Because of their common ancestry, similar organization and coordinate expression it has often been assumed that regulation of the globin gene clusters by HS -40 and the beta-LCR occurs via similar mechanisms. Using interspecific hybrids containing chromosomes with naturally occurring deletions of HS -40 we have shown that, in contrast to the beta-LCR, this element exerts no discernible effect on long-range chromatin structure and in addition does not influence formation of DNase I hypersensitive sites at the alpha globin promoters. These differences in the behaviour of HS -40 and the beta-LCR may reflect their contrasting influence on gene expression in transgenic mice and may result from the differing requirements of these elements in their radically different, natural chromosomal environments; the alpha cluster lying within a region of constitutively 'open' chromatin and the beta cluster in a segment of chromatin which opens in a tissue-specific manner. Differences in the hierarchical control of the alpha and beta globin clusters may exemplify more general differences in the regulation of eukaryotic genes which lie in similar open or closed chromosomal regions.
Structure of the human 3-methyladenine DNA glycosylase gene and localization close to the 16p telomere.
We recently reported the presence of four genes lying between the human alpha-globin gene cluster and the telomere of the short arm of chromosome 16 (16p). We now report that one of these genes encodes 3-methyladenine DNA glycosylase, an enzyme important in the repair of DNA after damage by alkylating agents. The gene comprises five exons, representation of which differs in independently isolated cDNA clones. Although the gene is widely expressed, the abundance of its mRNA is considerably higher in a colon adenocarcinoma cell line (HT29) than in other cell lines that were tested. The major positive erythroid-specific regulatory element controlling alpha-globin gene expression lies equidistant between the promoters of the alpha-globin genes and the 3-methyladenine DNA glycosylase gene. Interestingly, in contrast to the alpha-globin genes, expression of the 3-methyladenine DNA glycosylase gene is not influenced by the regulatory element in the human erythroleukemia cell line K562.
Conservation of position and sequence of a novel, widely expressed gene containing the major human alpha-globin regulatory element.
We have determined the cDNA and genomic structure of a gene (-14 gene) that lies adjacent to the human alpha-globin cluster. Although it is expressed in a wide range of cell lines and tissues, a previously described erythroid-specific regulatory element that controls expression of the alpha-globin genes lies within intron 5 of this gene. Analysis of the -14 gene promoter shows that it is GC rich and associated with a constitutively expressed DNase 1 hypersensitive site; unlike the alpha-globin promoter, it does not contain a TATA or CCAAT box. These and other differences in promoter structure may explain why the erythroid regulatory element interacts specifically with the alpha-globin promoters and not the -14 gene promoter, which lies between the alpha promoters and their regulatory element. Interspecies comparisons demonstrate that the sequence and location of the -14 gene adjacent to the alpha cluster have been maintained since the bird/mammal divergence, 270 million years ago.
Generation of bivalent chromatin domains during cell fate decisions.
BACKGROUND: In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined. RESULTS: Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3. CONCLUSIONS: While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different.
Analysis of the human alpha-globin gene cluster in transgenic mice.
A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.
Role of upstream DNase I hypersensitive sites in the regulation of human alpha globin gene expression.
Erythroid-specific DNase 1 hypersensitive sites have been identified at the promoters of the human alpha-like genes and within the region from 4 to 40 kb upstream of the gene cluster. One of these sites, HS-40, has been shown previously to be the major regulator of tissue-specific alpha-globin gene expression. We have now examined the function of other hypersensitive sites by studying the expression in mouse erythroleukemia (MEL) cells of various fragments containing these sites attached to HS-40 and an alpha-globin gene. High level expression of the alpha gene was observed in all cases. When clones of MEL cells bearing a single copy of the alpha-globin gene fragments were examined, expression levels were similar to those of the endogenous mouse alpha genes and similar to MEL cells bearing beta gene constructs under the control of the beta-globin locus control region. However, there was no evidence that the additional hypersensitive sites increased the level of expression or conferred copy number dependence on the expression of a linked alpha gene in MEL cells.
C/EBPα and GATA-2 Mutations Induce Bilineage Acute Erythroid Leukemia through Transformation of a Neomorphic Neutrophil-Erythroid Progenitor.
Acute erythroid leukemia (AEL) commonly involves both myeloid and erythroid lineage transformation. However, the mutations that cause AEL and the cell(s) that sustain the bilineage leukemia phenotype remain unknown. We here show that combined biallelic Cebpa and Gata2 zinc finger-1 (ZnF1) mutations cooperatively induce bilineage AEL, and that the major leukemia-initiating cell (LIC) population has a neutrophil-monocyte progenitor (NMP) phenotype. In pre-leukemic NMPs Cebpa and Gata2 mutations synergize by increasing erythroid transcription factor (TF) expression and erythroid TF chromatin access, respectively, thereby installing ectopic erythroid potential. This erythroid-permissive chromatin conformation is retained in bilineage LICs. These results demonstrate that synergistic transcriptional and epigenetic reprogramming by leukemia-initiating mutations can generate neomorphic pre-leukemic progenitors, defining the lineage identity of the resulting leukemia.
Iron Chelation in Transfusion-Dependent Patients With Low- to Intermediate-1-Risk Myelodysplastic Syndromes: A Randomized Trial.
BACKGROUND: Iron chelation therapy (ICT) in patients with lower-risk myelodysplastic syndromes (MDS) has not been evaluated in randomized studies. OBJECTIVE: To evaluate event-free survival (EFS) and safety of ICT in iron-overloaded patients with low- or intermediate-1-risk MDS. DESIGN: Multicenter, randomized, double-blind, placebo-controlled trial (TELESTO). (ClinicalTrials.gov: NCT00940602). SETTING: 60 centers in 16 countries. PARTICIPANTS: 225 patients with serum ferritin levels greater than 2247 pmol/L; prior receipt of 15 to 75 packed red blood cell units; and no severe cardiac, liver, or renal abnormalities. INTERVENTION: Deferasirox dispersible tablets (10 to 40 mg/kg per day) (n = 149) or matching placebo (n = 76). MEASUREMENTS: The primary end point was EFS, defined as time from date of randomization to first documented nonfatal event (related to cardiac or liver dysfunction and transformation to acute myeloid leukemia) or death, whichever occurred first. RESULTS: Median time on treatment was 1.6 years (interquartile range [IQR], 0.5 to 3.1 years) in the deferasirox group and 1.0 year (IQR, 0.6 to 2.0 years) in the placebo group. Median EFS was prolonged by approximately 1 year with deferasirox versus placebo (3.9 years [95% CI, 3.2 to 4.3 years] vs. 3.0 years [CI, 2.2 to 3.7 years], respectively; hazard ratio, 0.64 [CI, 0.42 to 0.96]). Adverse events occurred in 97.3% of deferasirox recipients and 90.8% of placebo recipients. Exposure-adjusted incidence rates of adverse events (≥15 events per 100 patient treatment-years) in deferasirox versus placebo recipients, respectively, were 24.7 versus 23.9 for diarrhea, 21.8 versus 18.7 for pyrexia, 16.7 versus 22.7 for upper respiratory tract infection, and 15.9 versus 0.9 for increased serum creatinine concentration. LIMITATIONS: The protocol was amended from a phase 3 to a phase 2 study, with a reduced target sample size from 630 to 210 participants. There was differential follow-up between treatment groups. CONCLUSION: The findings support ICT in iron-overloaded patients with low- to intermediate-1-risk MDS, with longer EFS compared with placebo and a clinically manageable safety profile. Therefore, ICT may be considered in these patients. PRIMARY FUNDING SOURCE: Novartis Pharma AG.
Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting.
Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (Rpearson > 0·91, P 0·03% LSC) from LSClow (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification.
Transient abnormal myelopoiesis without constitutional Down syndrome
Transient abnormal myelopoiesis (TAM) is a unique entity that usually occurs in children with Down syndrome (DS) or with trisomy 21 mosaicism. The somatic GATA1 mutation is a distinct feature of TAM. At presentation, TAM can resemble congenital leukemia (CL), which unlike TAM has an extremely poor prognosis and requires prompt therapeutic interventions. Therefore, correct and timely distinction between the two entities is crucial. We report a case of a phenotypically normal infant diagnosed with CL during the first weeks of life that retrospectively was reassessed as TAM. No acute myeloid leukemia (AML) specific mutations were found except for trisomy 21 confined exclusively to leukemic blasts. Retrospectively GATA1 mutation was also detected in malignant cells, but somatic genome appeared to be intact.
Human erythroleukemia genetics and transcriptomes identify master transcription factors as functional disease drivers.
Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.