Diversität der freilebenden und Partikel-assoziierten prokaryotischen Lebensgemeinschaften in der Ionischen See, Griechenland

Das Seegebiet über dem Calypso Deep (5189 m) in der Ionischen See wurde auf die Eignung als Standort eines Kubikkilometer großen Neutrino-Teleskops (KM3NeT) untersucht. Hierbei wurden die Hydrologie des Seegebiets und die prokaryotische Diversität bestimmt. Ein Fokus lag auf dem Nachweis von biolumineszenten und biokorrosiven Bakterien. Darüber hinaus konnten die Variationen der prokaryotischen Diversität in der Tiefe, unabhängig von der Temperatur, ermittelt werden. Die Hydrologie des Untersuchungsgebiets ist relativ konstant. Die Temperatur und die Salinität zeigen nur im Epipelagial eine klare Saisonalität im gewählten Probennahmenintervall. Die Konzentrationen der Nährstoffe, des partikulären organischen Kohlenstoffs und Stickstoffs sowie die Bakteriendichte zeigen, dass das Untersuchungsgebiet extrem oligotroph und in neritisch und ozeanisch unterscheidbar ist. Im Bathypelagial weisen die genannten Parameter konstant sehr geringe Werte auf. Das Vorkommen biolumineszenter Bakterien nur an Station N1 im Frühjahr 2008 als auch die im Verbundprojekt Innofond Schleswig-Holstein erstellte Arbeit zum trophie-abhängigen Vorkommen der [NiFe] Hydrogenasen unterstützen diese Klassifizierung des Gewässers (Barz et al., 2009). Die prokaryotische Diversität lässt eine tiefenzonierte Einteilung erkennen. Die Vorkommen tiefenspezifischer „operational taxonomic units“ (OTU) sind für die freilebende bakterielle Gemeinschaft in jeder Tiefe hoch (ca. 60 – 70 %). Ubiquitär detektiertbare OTU stellen einen ca. 20 % Anteil. In dem untersuchten Seegebiet bleibt die Artenvielfalt der freilebenden Gemeinschaft genauso wie die Temperatur über die Tiefe konstant. Die Diversität der freilebenden Bakterien wird, basierend auf den in dieser Arbeit erhobenen Daten, durch den Faktor Temperatur stärker beeinflusst als durch hohen hydrostatischen Druck. Hinzu kommt die teilweise Substratabhängigkeit der freilebenden Gemeinschaft von der, das organische Material remineralisierende, Partikel-assozierten Gemeinschaft. Die Diversität der Partikel-assoziierten Bakteriengemeinschaft nimmt über die Tiefe ab und wird wahrscheinlich von der Qualität des sedimentierenden organischen Materials bestimmt. Von den Archaea sind die Crenarchaea im gesamten Freiwasser dominant. Im Bereich des “deep chlorophyll maximum“ (DCM), evtl. im gesamten Mesopelagial, kommen Ammonium-oxidierende Crenarchaea vor, die einen wesentlichen Teil der lichtunabhängigen Primärproduktion leisten und die oberen Schichten mit Nitrit bzw. Nitrat versorgen. Im DCM sind einige Euryarchaea der Gruppe II Partikel-assoziierte. Euryarchaea der Gruppe III sind in dieser Studie zum ersten Mal als partikel-assoziiert identifiziert worden. Man findet sie nur in Tiefen des DCM, somit scheinen sie eng an die Primärproduktion gekoppelt zu sein. Das Wasser des Untersuchungsgebiets besitzt aufgrund der oben genannten Charakteristika eine hohe optische Güte. Einige biokorrosive Bakterien wurden erkannt und sollten bei der Materialauswahl berücksichtigt werden. Aus hydrologischer und biologischer Sicht kann der Standort für den Betrieb eines Neutrino-Teleskops als geeignet eingestuft werden.The Calypso Deep (5189 m), known as the deepest site in the Mediterranean Sea was verified for the acceptability of maintaining a cubic kilometer sized deep-sea neutrino telescope (KM3NeT). Our investigations focused on the analysis of the prokaryotic diversity in respect to prokaryotic bioluminescence and biocorrosion. Moreover, due to the rather high and constant temperature (>13 °C) of water even in the bathypelagial the study area is suitable for investigating the variation of the prokaryotic diversity over depth. The hydrology is quite constant at the study site. The sea surface temperature and the surface salinity (< 100 m) exhibit seasonal variations, solely. According to the nutrient and particulate organic carbon/nitrogen concentrations as well as to the very low prokaryotic bulk abundance found in the study area, it can be classified as extremely oligotrophic and may be divided into a neritic and an oceanic region. This categorisation is supported by the absence of [NiFe] hydrogenases in the complete study area and by the seasonal incidence of bioluminescene genes only in coastal waters. The presence of [NiFe] hydrogenases is restricted to eutrophic habitats. The prokaryotic diversity is stratified throughout the water column and shows distinct phylogenetic depth specificity. Approximately 20 % of the detected operational taxonomic units (OUT) are ubiquitous. A compositional change of the diversity but no decrease in over all diversity with depth was observed. These findings might result from the constant temperature observed between surface and bottom rather than on hydrostatic pressure alterations. Thus, the prokaryotic diversity could be mainly controlled by temperature. The diversity of the particle-associated community decreases with depth. Likely, its quality and consecutively the quality of the free-living community are determined by the character of the particulate organic matter. The crenarchaea constitute the majority of the free-living archaeal community at all depth. In contrast, the group II and III euryarchaea are dominant in the particle-associated fraction, only. Group III euryarchaea are restricted to the “deep chlorophyll maximum” (DCM) and particles which point to a possible interconnection with the primary production. At depth of the DCM a second kind of primary production was observed. Ammonium-oxidizing crenarchaea (N. maritimus) were detectable at 100 m depth providing nitrite for nitrifying bacteria, supporting an efficient nutrient remineralisation which is essential for the trophy status of extreme oligotrophic habitats. Summing up these results and adding the seasonal invariability of the analyzed parameters in the bathypelagial, the aquatic system at the study site exhibits excellent optical quality. Although biocorrosive species were found, the study site is favourable for installing a neutrino telescope

Distribution Analysis of Hydrogenases in Surface Waters of Marine and Freshwater Environments

Background Surface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H2 evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H2 metabolism in marine environments is nitrogen fixation. Principal Findings We analyzed marine and freshwater environments for the presence and distribution of genes of all known hydrogenases, the enzymes involved in biological hydrogen turnover. The total genomes and the available marine metagenome datasets were searched for hydrogenase sequences. Furthermore, we isolated DNA from samples from the North Atlantic, Mediterranean Sea, North Sea, Baltic Sea, and two fresh water lakes and amplified and sequenced part of the gene encoding the bidirectional NAD(P)-linked hydrogenase. In 21% of all marine heterotrophic bacterial genomes from surface waters, one or several hydrogenase genes were found, with the membrane-bound H2 uptake hydrogenase being the most widespread. A clear bias of hydrogenases to environments with terrestrial influence was found. This is exemplified by the cyanobacterial bidirectional NAD(P)-linked hydrogenase that was found in freshwater and coastal areas but not in the open ocean. Significance This study shows that hydrogenases are surprisingly abundant in marine environments. Due to its ecological distribution the primary function of the bidirectional NAD(P)-linked hydrogenase seems to be fermentative hydrogen evolution. Moreover, our data suggests that marine surface waters could be an interesting source of oxygen-resistant uptake hydrogenases. The respective genes occur in coastal as well as open ocean habitats and we presume that they are used as additional energy scavenging devices in otherwise nutrient limited environments. The membrane-bound H2-evolving hydrogenases might be useful as marker for bacteria living inside of marine snow particles

Sequential Evolution Versus a Single Catastrophic Event (Chromothripsis) in the Pathogenesis of AML with Complex Aberrant Karyotype

Abstract Abstract 517 Background: In AML, the concept of gradual evolution through a sequence of genetic alterations and clonal expansion was favored thus far, but has been recently challenged by a hypothesis that one catastrophic event generates multiple lesions across the genome in a single step. The term “chromothripsis” was introduced for a single catastophic event leading to the shattering of a single chromosome followed by rejoining and thereby resulting in a highly recombined chromosome (Stephens PJ et al., Cell 2011). Aim: We addressed the question whether AML with complex karyotype - defined as 4 or more abnormalities - evolves by sequential gradual acquisition of chromosome abnormalities or by a single catastrophic event. Patients and Methods: We selected 889 AML cases (de novo: n=634, secondary AML: n=164, therapy-related AML: n=91) presenting a complex karyotype at diagnosis. These were analyzed by chromosome banding analysis, 24-color-FISH, interphase-FISH, array CGH (n=78) and TP53 mutation analysis (n=195). Results: In 518/889 (58.3%) cases at least one subclone was observed that showed extra chromosome aberrations, thus demonstrating clonal evolution already at the timepoint of AML diagnosis. Within these, 77/518 (14.9%) cases showed a primary clone with only one cytogenetic abnormality. Two of these were recurrent single abnormalities: del(5q) (n=62), +8 (n=4). Clonal evolution was more frequent in cases with del(5q) as compared to those without (404/666 (60.7%) vs 117/223 (52.5%); p=0.034) while no association with loss of 7q, loss of 17p, TP53 mutation or type of AML (de novo vs secondary vs therapy-related AML) was observed. In 46 cases which evolved from MDS (n=43) or MPN (n=3) chromosome banding analysis had been performed prior to the diagnosis of AML. In 21/46 (45.7%) cases karyotype had not changed while in 25/46 (54.3%) cases clonal evolution had occurred. 57 cases were analyzed at relapse of AML; in 28 (49.1%) cases clonal evolution was detected. Additionally, 78/889 cases were evaluated by array CGH. The occurrence of chromothripsis was analyzed following the definition by Rausch et al. (Cell 2012) with at least 10 segmental copy-number changes involving two or three distinct copy-number states on a single chromosome. Evidence of at least one “shattered” chromosome was found in 24/78 (30.8%) cases. In 21 cases only one chromosome fulfilled these criteria, while in 3 cases chromothripsis affected two or more chromosomes. The chromosome most frequently affected by “shattering” was chromosome 11, observed in 18 (85.7%) cases, followed by chromosomes 2 and 21, which were affected in 2 cases each. Chromosomes 1, 5, 7, 13, 15, 16 and 20 showed signs of chromothripsis in single cases only. In 19/24 (79.2%) cases showing evidence of chromothripsis a high level amplification was observed for the MLL gene (11q23) in 17 cases and for the ERG gene (21q22) in 2 cases. Thus, amplifications were more frequent than in cases without chromothripsis (21/54, 38.89%; p=0.001), while no association was observed between chromothripsis and deletions of 5q, 7q or 17p or TP53 mut, presence of clonal evolution or type of AML (de novo vs secondary vs therapy-related AML). With respect to outcome within the subgroup of AML with complex karyotype only TP53 mutations and the presence of 5q deletions were significantly associated with overall survival (relative risk (RR) for shorter OS in TP53 mut cases: 3.19, p&lt;0.0001, and in del(5q) cases: 1.61, p=0.006; median OS in TP53mut vs TP53wt cases: 4.6 vs 22.0 months, p&lt;0.0001; median OS in del(5q) vs non-del(5q) cases: 5.7 vs 14.4 months, p=0.006), while presence of deletions of 7q, or 17p, chromothripsis and clonal evolution showed no impact on outcome. In multivariable Cox regression analysis only TP53mut had an independent association with shorter OS (RR: 3.12, p=0.001). Conclusions: 1. In AML with complex karyotype harboring a 5q deletion the acquisition of additional abnormalities is more frequent than in cases without del(5q). 2. Chromothripsis does occur in AML with complex karyotype. However, the “shattering” of one chromosome was never observed as the sole abnormality, indicating that chromothripsis and sequential genetic evolution are not alternative but more likely combined mechanisms in AML. 3. Our data demonstrates that stepwise clonal evolution is more frequent than chromothripsis and thus likely to be more important in the pathogenesis of AML with complex karyotype. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. </jats:sec

Gene Amplifications In 84 Patients With Acute Myeloid Leukemia and 31 Patients With Myelodysplastic Syndrome Investigated By Array CGH

Abstract Background Gains and losses of chromosomal material are frequent in AML and MDS and usually lead to loss or gain of a single copy of a whole chromosome, a chromosome arm or small stretches of the chromosome that may be microscopically invisible. More rarely, amplifications of chromosomal regions (defined as the presence of more than 6 copies of a region per cell) are observed. These supernumerary copies are located either extrachromosomally as small acentric chromosomal structures - so called double-minutes (dmin) - or intrachromosomally as large contiguous stretches of amplified DNA, so called homogeneously staining regions (HSR). Aims Characterize AML and MDS cases with gene amplifications with respect to size, affected genes and accompanying chromosomal abnormalities as well as TP53 status. Patients and Methods 84 AML and 31 MDS cases with cytogenetically visible amplifications were selected for this study. All cases were analyzed by array CGH, chromosome banding analysis, sequencing for TP53 mutations as well as FISH for TP53 deletions. Results The cohort comprised 55 (47.8%) males and 60 (52.2%) females with a median age of 72.0 years (range 38.0 - 90.3 years). A complex karyotype (≥4 aberrations) was present in 92/115 (80.0%) cases (AML=65/84 (77.4%); MDS=27/31 (87.1%)). In total, 385 amplified regions were identified by array CGH. In more detail: 3q26 (AML: n=6; MDS: n=3), 8q24 (AML: n=15; MDS: n=1), 11q21-25 (AML: n=42; MDS: n=13), 13q12 (AML: n=3; MDS: n=1), 13q31 (AML: n=3; MDS: n=2), 19p13 (AML: n=2; MDS: n=4), and 21q21-q22 (AML: n=24; MDS: n=5). The median number of amplified regions was 3 (range 1-18). In 14/115 (12.2%) cases, the amplification was located in dmins (AML: n=11; MDS: n=3) and in 101/115 (87.8%) patients in HSR (AML: n=73; MDS: n=28). In 40 of the latter 101 cases (39.6%) (AML: n=24; MDS: n=16) the amplification was located on a ring chromosome (rc). In patients with complex karyotypes we detected a significantly higher number of amplified regions as compared to non-complex karyotypes (3.5 vs. 2.8; p=0.015). No association between the complexity of the karyotype and the structural type of the amplification (dmin vs rc) was observed. Cases with non-complex karyotypes frequently harbored a 5q deletion (6/23; 26.1%) or chromosome 8 abnormalities (3/23; 13.0%). Within the subgroup of non-complex karyotypes del(5q) cases showed a tendency to a higher number of amplified regions (3.6 vs. 1.9; p=0.140). Further, amplifications of 11q genes were more frequent in complex karyotypes (54.4% vs. 21.7%; p=0.005), whereas 8q amplifications were more frequent in non-complex karyotypes (43.5% vs. 4.4%; p&lt;0.001). We detected a large region on band 11q24, which was amplified in 41/53 (77.4%) cases. This commonly amplified region contains 1,575 genes including the MLL gene. Cases harboring dmins had shorter amplified regions compared to cases with rc (4,428,112.5 bp vs. 18,265,496.9 bp; p=0.028). Moreover, we detected a positive correlation of patients having a rc and gene amplification on chromosome 11q23-25 (p&lt;0.05). On chromosome 3q, 8/9 (88.9%) cases shared a minimal amplified region covering the EVI1 gene. In comparison to samples obtained from healthy donors (n=47), the EVI1 expression was significantly higher in cases with EVI1 amplification (87.4 vs. 0.5; p=0.048). On chromosome 21q the regions of amplifications were heterogeneous. However, we detected a minimal region containing 11 genes including ERG which was amplified in 26/29 (89.7%) patients. ERG expression data was available in 8 cases and was significantly higher compared to a control cohort of AML with normal karyotype (n=331) (729.2 vs. 229.0; p=0.05). On chromosome 8 an amplified region was identified in 15/16 cases. In 14 of these cases (87.5%) the region included MYC. TP53mut were present in 93/115 (80.9%) patients, accompanied by a TP53del in 28/93 (30.1%) cases. Interestingly, cases harboring a TP53mut had more amplified regions compared to TP53wt (3.4 vs. 1.7; p&lt;0.001). Conclusions 1. MLL is the most frequently amplified gene in AML and MDS. 2. Patients with complex karyotypes or TP53mut harbored more amplified regions compared to patients with non-complex karyotypes and TP53wt. 3. Amplifications on 11q were more frequent in complex karyotype whereas gene amplifications on 8q were predominantly observed in non-complex karyotypes. 4. EVI1 and ERG gene amplifications lead to a higher expression of the respective genes. Disclosures: Roller: MLL Munich Leukemia Laboratory: Employment. Weber:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. </jats:sec

Array CGH Identifies Copy Number Changes In 10% Of 520 MDS Patients With Normal Karyotype: Deletions Encompass The Genes TET2, DNMT3A, ETV6, NF1, RUNX1, and STAG2 and Are Associated With Shorter Survival

Abstract Background In MDS, cytogenetic aberrations play an important role for classification and prognostication. The original IPSS and the revised IPSS classifiers have clearly demonstrated the prognostic impact of distinct cytogenetic abnormalities. The vast majority of chromosome aberrations in MDS are gains or losses of chromosomal material while balanced rearrangements are rare. However, more than 50% of MDS and even more in low risk MDS harbor a normal karyotype. Chromosome banding analysis can only detect gains and losses of more than 10 Mb size due to its limited resolution and is dependent on proliferation of the MDS clone in vitro to obtain metaphases. Array CGH has a considerably higher resolution and does not rely on proliferating cells. Aims In this study we addressed the question whether MDS with normal karyotype harbor cytogenetically cryptic gains and losses. Patients and Methods 520 MDS patients with normal karyotype were analyzed by array CGH (Human CGH 12x270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). For all patients cytomorphology and chromosome banding analysis had been performed in our laboratory. The cohort comprised the following MDS subtypes: RA (n=22), RARS (n=43), RARS-T (n=27), RCMD (n=124), RCMD-RS (n=111), RAEB-1 (n=104), and RAEB-2 (n=89). Median age was 72.2 years (range: 8.9-90.1 years). Subsequently, recurrently deleted regions detected by array CGH were validated using interphase-FISH. Results In 52/520 (10.0%) patients copy number changes were identified by array CGH. Only eight cases (1.5%) harbored large copy number alterations &gt;10 Mb in size, as such generally detectable by chromosome banding analysis. These copy number alterations were confirmed by interphase-FISH. They were missed by chromosome banding analysis due to small clone size (n=2), insufficient in vitro proliferation (n=3) or poor chromosome morphology (n=3). In the other 44 patients with submicroscopic copy number alterations 18 gains and 32 losses were detected. The sizes ranged from 193,879 bp to 1,690,880 bp (median: 960,176 bp) in gained regions and 135,309 bp to 3,468,165 bp (median: 850,803 bp) in lost regions. Recurrently deleted regions as confirmed by interphase-FISH encompassed the genes TET2 (4q24; n=9), DNMT3A (2p23; n=3), ETV6 (12p13; n=2), NF1 (17q11; n=2), RUNX1 (21q22; n=2), and STAG2 (Xq25, deleted in 2 female patients). No recurrent submicroscopic gain was detected. In addition, we performed survival analysis and compared the outcome of patients with normal karyotype also proven by array CGH (n=462) to patients with aberrant karyotype as demonstrated by array CGH (n=52). No differences in overall survival were observed. However, overall survival in 35 patients harboring deletions detected solely by array CGH was significantly shorter compared to all others (median OS: 62.1 vs 42.4 months, p=0.023). Conclusions 1. Array CGH detected copy number changes in 10.0% of MDS patients with cytogenetically normal karyotype as investigated by the gold standard method, i.e. chromosome banding analysis. 2. Most of these alterations were submicroscopic deletions encompassing the genes TET2, ETV6, DNMT3A, NF1, RUNX1, and STAG2. 3. Interphase-FISH for these loci can reliably pick up these alterations and is an option to be easily performed in routine diagnostics in MDS with normal karyotype. 4. Patients harboring deletions detected solely by array-CGH showed worse prognosis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Raitner:MLL Munich Leukemia Laboratory: Employment. Holzwarth:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. </jats:sec

Acute Erythroid Leukemia (AEL) Can Be Separated Into Distinct Prognostic Subsets Based On Cytogenetic and Molecular Genetic Characteristics

Abstract Abstract 1394 Background: Acute erythroid leukemia (AEL) is characterized by a predominant erythroid population and is comprising &lt;5% of adult AML cases. Because of the relative rarity of AEL, few large studies have examined underlying clinical and genetic features. Aims: Molecular and cytogenetic characterization of AEL and identification of genes with prognostic impact. Patients and Methods: We studied an unselected cohort of 94 AEL patients including 32 female and 62 male cases; median age was 69.0 yrs (range: 21.3–88.3 yrs). Survival data was available in 73 cases; median survival was 15.9 months. First, chromosome banding analysis (n=94) was performed. In addition, all cases with normal karyotype (NK) were investigated by CGH arrays (n=32) (Human CGH 12×270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). Further, mutation screening for ASXL1 (n=87), CEBPA (n=94), DNMT3A (n=94), FLT3 (both internal tandem duplication (ITD) (n=93), and tyrosine-kinase domain (TKD) mutations (n=85)), IDH1 (n=93), IDH2 (n=65), NRAS (n=91), KRAS (n=93), MLL-PTD (n=79), NPM1 (n=94), RUNX1 (n=94), TP53 (n=94), and WT1 (n=90) was performed by 454 amplicon deep-sequencing (Roche, Branford, CT), Sanger sequencing or melting curve analyses. CGH array data analysis was performed using Nexus Copy Number 6.0 (BioDiscovery Inc, El Segundo, CA). Results: Cytogenetic data was available for all cases: 48 cases (51.1%) presented an intermediate-risk and 46 (48.9%) cases an unfavorable cytogenetic category according to the MRC Classification. By CGH array analysis 30/32 cases retained a NK, whereas in two cases small aberrations were detected: case 1: deletion of the CEBPA gene, case 2: duplication 11q13.3 to 11q25 including the ATM and MLL gene. Molecular mutations were detected in 85/94 patients (90.4%). 63.5% (54/85) of mutated patients carried one, whereas 36.5% (31/85) of cases harbored two (n=22) or more (n=9) mutations. In detail, TP53 was the most frequently mutated gene (41 cases, 43.6%). Other alterations were detected in NPM1 (15/94; 16.0%); DNMT3A (12/94; 12.8%); ASXL1 (8/87; 9.2%); MLL-PTD (7/79; 8.9%); RUNX1 (8/94; 8.5%); IDH1 (6/93; 6.5%); WT1 (5/90; 5.6%); IDH2 (3/65; 4.6%); NRAS (3/91; 3.3%); KRAS (3/93; 3.2%); FLT3-ITD (3/93, 3.2%), FLT3-TKD (3/85, 3.5%), and CEBPA (1/94). First, we were interested in any correlation with the respective karyotype and observed that NPM1, RUNX1, and WT1 mutations correlated with an intermediate-risk karyotype (NPM1: 15/48 vs 0/46, P&lt;0.001; RUNX1: 8/48 vs 0/46, P=0.006; WT1: 5/46 vs 0/44, P=0.056), whereas TP53mut correlated with the unfavorable karyotype (38/46 vs 3/48, P&lt;0.001). Within the cytogenetically adverse subset TP53mut were associated with complex karyotype (36/38 vs 2/8, P&lt;0.001). In addition, NPM1mut correlated with lower age (56±15 vs 67±13 yrs, P=0.002), whereas mutations in ASXL1, DNMT3A, and TP53 correlated with higher age (73±4 vs 64±15, P=0.001; 71±6 vs 65±14, P=0.015; 71±8 vs 61±15, P&lt;0.001). NPM1mut were associated with longer, and RUNX1mut and TP53mut with shorter OS (OS after 2 yrs: NPM1mut vs wt: 85.1% vs 28.3%, P=0.001; RUNX1mut vs wt: 0% vs 45.2%, P=0.007; TP53mut vs wt: 9.4% vs 61.6%, P=0.001). In the univariable Cox regression analyses mutations in NPM1 (HR 0.12; P=0.004), RUNX1 (HR 3.99; P=0.013), TP53 (HR 3.19; P=0.001), age (HR 4.24, P=0.001) and adverse cytogenetics (HR 2.98, P=0.002) were significantly associated with OS. Independent prognostic factors in multivariable Cox regression analysis were: age (HR 2.6, P=0.047) and RUNX1mut (HR 6.3, P=0.006). Of note, when separating MRC intermediate from MRC adverse cases, we confirmed the longer OS of NPM1 and shorter OS of RUNX1 mutated cases in comparison to NPM1, RUNX1 wt cases (OS after 2 yrs: NPM1mut vs wt: 85.1% vs 46.3%, P=0.027; RUNX1mut vs wt: 0% vs 69.0%, P&lt;0.001). Conclusions: (1) The frequency of cases with complex or other adverse karyotypes within the AEL cohort is very high (48.9%), (2) 93.7% of cases with NK also showed a NK using high-resolution CGH arrays. (3) Overall, a remarkably high mutation frequency of 90.4% was found. (4) NPM1 and RUNX1mut were exclusively detected in the cytogenetically intermediate-risk MRC, TP53 mut predominantly in the MRC adverse group and mainly in cases with complex karyotype. (5) In addition to chromosome banding analysis mutation screening of RUNX1 and NPM1 in cases with intermediate-risk karyotype should be considered for better prognostication. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Bacher:MLL Munich Leukemia Laboratory: Employment. Poetzinger:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. </jats:sec

Activation and repression by oncogenic MYC shape tumour-specific gene expression profiles

In mammalian cells, the MYC oncoprotein binds to thousands of promoters(1-4). During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes(1). In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up-and downregulated genes that have considerable prognostic value(5-7). To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation(8). Third, complex formation with MIZ1 (also known as ZBTB17)(9) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response