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Background
Somatic mutations identified in patients with myelodysplastic syndromes (MDS) are associated with disease features and carry prognostic information independent of the International ...Prognostic Scoring System (IPSS) and the revised IPSS (IPSS-R). Risk models that include mutation information have been proposed, but not widely adopted. In practice, there is no consensus on how to best combine clinical information with tumor sequencing data to predict prognosis. To accomplish this, we must define the relevant genes to consider and accurately measure their prognostic impact. Here we examine the relationship between mutations in MDS-associated genes and clinically relevant measures, including overall survival, in a large, multi-center analysis of MDS patient cohorts collected around the globe.
Methods
Data on 3392 MDS patients gathered by members of the International Working Group for Prognosis in MDS-Molecular Committee were combined under the aegis of the MDS Foundation. Patients gave informed consent for collection of their data and tumor samples at their respective institutions in accordance with the Declaration of Helsinki. Samples were examined for somatic mutations primarily by next generation sequencing. Categorical variables were compared using a chi-squared test, while continuous variables were compared using a Wilcoxon rank-sum test. Overall survival (OS) was calculated from the date of the sequenced sample to the date of death and was censored at transplant or the last known follow-up time. P-values are two-sided and considered significant at the <0.001 level to adjust for multiple comparisons.
Results
Survival data were available for 3200 patients with a median follow up of 3.7 years and included 1671 deaths. Median survival of the cohort was 2.88 years. The 27 genes sequenced in at least half of the cohort and mutated in > 1.5% of samples were included for analysis (Figure 1). Mutations in 12 genes were strongly associated with shorter OS in univariate analyses (p<0.001 for each gene): ASXL1, CBL, EZH2, IDH2, NF1, NRAS, PTPN11, RUNX1, SRSF2, STAG2, TP53, and U2AF1. Only mutations of SF3B1 were associated with a longer OS at this significance threshold. The large size of the cohort allowed for more precise estimates of survival in less frequently mutated genes. For example, mutations of IDH2 (present in 3.4% of cases, n=103) were associated with shorter OS (hazard ratio HR 1.61, 95% confidence interval CI 1.26-2.05; p=0.0001) whereas IDH1 mutations (present in 2.4% of cases, n=77) were only marginal (HR 1.29, CI: 0.97-1.72; p=0.082), demonstrating the distinct impact of mutations in these highly related genes.
IPSS-R risk groups could be determined for 2173 patients and were strongly associated with OS. Adjusting the hazard ratio of death for IPSS-R risk groups identified several mutated genes with independent prognostic significance: TP53 (HR 2.37, CI 1.94-2.90), CBL (HR 1.57, CI 1.22-2.03), EZH2 (HR 1.55, CI 1.22-2.03), and RUNX1 (HR 1.50, CI 1.24-1.83). Mutations of U2AF1 (HR 1.29, CI 1.06-1.58) and ASXL1 (HR 1.21, CI 1.04-1.41) retained a more modest association with shorter OS. Adjustment for IPSS-R risk groups also moderated the favorable risk associated with mutations of SF3B1 (HR 0.83, CI 0.70-0.99). Patients without mutations in any of the 6 adverse genes above represented 58% of the fully sequenced cohort and had a longer median survival than patients with adverse mutations (4.8 years vs. 1.6 years respectively, p < 0.0001; Figure 2) even after correction for IPSS-R risk groups (adjusted HR 0.59, CI 0.51-0.67).
Multivariable analysis of this dataset will examine the combined contribution of mutated genes to prognosis. A mutation score based on survival risk will be proposed and internally validated. The impact of somatic mutation in patients traditionally considered lower risk will be explored.
Conclusions
This large study definitively validates the prognostic value of mutations in several MDS-associated genes while clarifying the significance of other, less frequently mutated ones. Mutations in several genes retain their prognostic significance after adjustment for IPSS-R risk groups, indicating that these select abnormalities could refine the prediction of prognosis when incorporated into a clinical scoring system such as the IPSS-RM. The results of this analysis will serve as the template with which to build an integrated molecular risk model for MDS.
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Bejar:Alexion: Other: ad hoc advisory board; Celgene: Consultancy, Honoraria; Genoptix Medical Laboratory: Consultancy, Honoraria, Patents & Royalties: MDS prognostic gene signature. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Fenaux:Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Shih:Novartis: Research Funding. Komrokji:Celgene: Consultancy, Research Funding; Incyte: Consultancy; Novartis: Research Funding, Speakers Bureau; Pharmacylics: Speakers Bureau. List:Celgene Corporation: Honoraria, Research Funding. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. Campbell:14M genomics: Other: Co-founder and consultant. Ebert:Celgene: Consultancy; Genoptix: Consultancy, Patents & Royalties; H3 Biomedicine: Consultancy.
Hematopoietic stem cells are important clinically, both as targets of disease and as reagents for cellular therapy. Studies in hematopoietic stem cell biology have been hampered by difficulties in ...purifying and manipulating these cells. To facilitate these studies, we sought to develop a system for targeting genes of interest to the hematopoietic stem cell compartment in transgenic mice.
We used Sca-1, a glycosyl phosphatidylinositol-anchored protein expressed on the surface of all hematopoietic stem cells in commonly used inbred mouse strains. We created a mutant
Sca-1 allele in which the enhanced green fluorescent protein (EGFP) cDNA is integrated into the
Sca-1 locus by homologous recombination in embryonic stem cells.
EGFP protein is detectable in all hematopoietic tissues of mice heterozygous for the mutant
Sca-1 allele. Growth and development of these mice are normal. No adverse effects of long-term, high-level EGFP expression were noted. Sca-1 positive cells coexpress EGFP in all tissues and lineages examined, as predicted by the targeting strategy. Sca-1 and EGFP expression are coordinately up-regulated in splenocytes from mutant mice. The Lin
−
EGFP
+
bone marrow population contains all progenitor activity in
Sca-1
+
/EGFP mice. The Lin
−
EGFP
+
bone marrow cells are equivalent to Lin
−Sca-1
+ cells in long-term repopulation and serial transplantation assays.
The hematopoietic stem cell compartment appears to be targeted in
Sca-1
+
/EGFP mutant mice. This system should be useful for studying the normal biology of hematopoietic stem cells and for targeting other genes to this cellular compartment.
The t(8;21)(q22;q22) translocation, present in 10-15% of acute myeloid leukemia (AML) casesresults in the production of the AML1/ETO fusion protein. Expression of AML1/ETO in patientsor mouse models ...is not sufficient to induce AML. Despite convincing evidence that AML1/ETOis directly involved in the pathogenesis of AML, the underlying mechanism is not wellunderstood. Genetic and biochemical experiments suggest that AML1/ETO is a dominantinhibitor of the core binding factor (CBF) transcription complex that includes AML1 (RUNX1), theN-terminal fusion partner in the t(8;21). We generated and recently characterized a novel strainof transgenic mice in which the AML1/ETO cDNA was inserted into the Ly-6A gene thatencodes Sca1, a well-characterized marker of murine hematopoietic stem cells. Unexpectedly,transgene expression assessed by flow cytometry was significantly lower than predicted inlymphocytes from these mice. We have confirmed this finding at the mRNA level and suggestthat this phenotype is a consequence of dominant inhibition of transgene expression byAML1/ETO. The dominant negative characteristics of AML1/ETO may be important for AMLpathogenesis and may provide a molecular target for therapeutic intervention.
Genome-wide mapping approaches are needed to more fully understand the genetic basis of chemotherapy response. Because of technical and ethical limitations, cancer pharmacogenomics has not yet ...benefited from traditional robust familial genetic strategies. We have therefore explored the use of the inbred mouse as a genetic model system in which to study response to the cytotoxic agent cyclophosphamide. Multiple phenotypes have been assessed in response to cyclophosphamide in up to 19 inbred mouse strains, including in vitro hematopoietic progenitor cell toxicity and the mobilization of hematopoietic progenitor cells into peripheral blood. Hematopoietic progenitor cell toxicity in vitro varied 2-fold among strains, whereas in vivo progenitor cell mobilization varied almost 75-fold among strains. Males mobilized more hematopoietic progenitor cells than did females, and the low-mobilization phenotype was dominant to the high-mobilization phenotype in F1 hybrid animals. In an initial attempt to analyze candidate genes, genetic variation was assessed in three cytochrome P-450 genes involved in the metabolism of cyclophosphamide. Resequencing of eight strains identified 26 polymorphisms in these genes that may influence response to cyclophosphamide. Distinct regions of high- and low-polymorphism rates were identified, and two common haplotypes were shared among the strains for each gene that exhibited variation. This phenotypic and genotypic variation among inbred strains provides a framework for cyclophosphamide pharmacogenomic discovery.
In this pilot study, we used primary human acute myeloid leukemia (AML) cell genomes as templates for exonic PCR amplification, followed by high-throughput resequencing, analyzing ≈7 million base ...pairs of DNA from 140 AML samples and 48 controls. We identified six previously described, and seven previously undescribed sequence changes that may be relevant for AML pathogenesis. Because the sequencing templates were generated from primary AML cells, the technique favors the detection of mutations from the most dominant clones within the tumor cell mixture. This strategy represents a viable approach for the detection of potentially relevant, nonrandom mutations in primary human cancer cell genomes.
The random insertion of transgenes into the genomic DNA of mice usually leads to widely variable levels of expression in individual founder lines. To study the mechanisms that cause variegation, we ...designed a transgene that we expected to variegate, which consisted of a β-globin locus control region 5′ HS-2 linked in tandem to a tagged human β-globin gene (into which a Lac-Z cassette had been inserted). All tested founder lines exhibited red blood cell-specific expression, but levels of expression varied >1000-fold from the lowest to the highest expressing line. Most of the variation in levels of expression appeared to reflect differences in the percentage of cells in the peripheral blood that expressed the transgene, which ranged from 0.3% in the lowest expressing line to 88% in the highest; the level of transgene expression per cell varied no more than 10-fold from the lowest to the highest expressing line. These differences in expression levels could not be explained by the location of transgene integration, by an effect of β-galactosidase on red blood cell survival, by the half life of the β-galactosidase enzyme or by the age of the animals. The progeny of all early erythroid progenitors (BFU-E colony-forming cells) exhibited the same propensity to variegate in methylcellulose-based cultures, suggesting that the decision to variegate occurs after the BFU-E stage of erythroid differentiation. Collectively, these data suggest that variegation in levels of transgene expression are due to local, integration site-dependent phenomena that alter the probability that a transgene will be expressed in an appropriate cell; however, these local effects have a minimal impact on the transgene's activity in the cells that initiate transcription.
The Ly-6 family includes a number of highly homologous, low molecular weight glycophosphatidylinositol-linked proteins expressed on hematopoietic and lymphoid cells. The best characterized family ...member is Sca-1 (Ly-6A/E), an antigen commonly used for purification of murine pluripotent hematopoietic cells. We sought to characterize the genomic locus surrounding the Sca-1 gene. We identified several overlapping P1 artificial chromosomes containing the Sca-1 gene and mapped one of these to mouse chromosome 15D3.1-3.3, the region previously shown to contain members of the murine Ly-6 gene family. We then mapped this clone and found that the Sca-2 gene lies 35.4 kilobase (kb) downstream of Sca-1 in the opposite transcriptional orientation. This is the first direct demonstration of physical linkage of Ly-6 genes. A novel gene, highly homologous to Sca-1 was identified and localized 13.4 kb downstream of Sca-1. This gene, which we designated Ly-6M, shares several structural features conserved among members of the Ly-6 family. Ly-6M messenger RNA (mRNA) is easily detectable in hematopoietic tissue (bone marrow, spleen, thymus, peritoneal macrophages) as well as kidney and lung. No mRNA expression was detected in heart, stomach, liver, small intestine, brain, or skin. Ly-6M protein is detectable on 10% to 15% of peripheral blood leukocytes, including monocytes and a subpopulation of B220(+) cells. Ly-6M is broadly distributed in the bone marrow, with prominent expression on monocytes and myeloid precursors. The identification and characterization of Ly-6M adds a new member to a complex family of homologous, tightly linked genes that have proven extremely useful reagents for defining populations within the hematopoietic system.
Two recombinant retroviral systems are described that can be used to analyze antiretroviral drug activity and HIV breakthrough (replication in the presence of thedrug). The first system utilizes a ...recombinant HIV encoding beta-galactosidase as a reporter gene (HIVLacZ). The defective HIV-LacZ virus is produced in COS cells after co-transfection of a plasmid encoding the HIV-LacZ genome with a plasmid encoding HIV proteins necessary for packaging and infectivity. Subsequent infection of CD4+ target cells, followed by assay for LacZ expression, permits the rapid identification of individual virus-infected cells. This system can be used to quantitate the inhibition of early events in the HIV replicative cycle and is suitable for the screening of compounds for anti-HIV activity. However, this system cannot be used to analyze HIV drug resistance because of the limited genetic heterogeneity of the virus that is produced in COS cells. To circumvent this problem, a second system has been developed in which heterogenous recombinant HIV is produced by rescue with replication-competent ′helper′ HIV. This system required the production of CD4+ cell lines containing defective proviruses encoding either LacZ or guanosine phosphoribosyl transferase (gpt). The defective proviruses are rescued by infection of the cell lines with ′helper′ HIV and used to infect target cells in the presence of antiretroviral agents. Subsequent reporter gene assay is used to identify virus-infected cells. This system has been used to detect rare HIV breakthrough infection of cells in the presence of the non-nucleoside reverse transcriptase inhibitor TIBO R82150. Similar analyses with other antiretroviral agents, alone and in combination, may help identify therapeutic strategies that minimize breakthrough replication of HIV.
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