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The revised 2017 European LeukemiaNet (ELN) recommendations for genetic risk stratification of acute myeloid leukemia have been widely adopted, but have not yet been validated in large cohorts of AML patients. We studied 1116 newly diagnosed AML patients (age range, 18–86 years) who had received induction chemotherapy. Among 771 patients not selected by genetics, the ELN-2017 classification re-assigned 26.5% of patients into a more favorable or, more commonly, a more adverse-risk group compared with the ELN-2010 recommendations. Forty percent of the cohort, and 51% of patients ≥60 years, were classified as adverse-risk by ELN-2017. In 599 patients <60 years, estimated 5-year overall survival (OS) was 64% for ELN-2017 favorable, 42% for intermediate-risk and 20% for adverse-risk patients. Among 517 patients aged ≥60 years, corresponding 5-year OS rates were 37, 16, and 6%. Patients with biallelic CEBPA mutations or inv(16) had particularly favorable outcomes, while patients with mutated TP53 and a complex karyotype had especially poor prognosis. DNMT3A mutations associated with inferior OS within each ELN-2017 risk group. Our results validate the prognostic significance of the revised ELN-2017 risk classification in AML patients receiving induction chemotherapy across a broad age range. Further refinement of the ELN-2017 risk classification is possible.

Several studies demonstrated a gene-density-correlated radial organization of chromosome territories (CTs) in spherically shaped nuclei of human lymphocytes or lymphoblastoid cells, while CT arrangements in flat-ellipsoidal nuclei of human fibroblasts are affected by both gene density and chromosome size. In the present study, we performed fluorescence in situ hybridization (FISH) experiments to three-dimensionally preserved nuclei (3D-FISH) from human and nonhuman primate cultured lymphoblastoid cells and fibroblasts. We investigated apes, Old, and New World monkeys showing either evolutionarily conserved karyotypes, multiple translocations, fusions, or serial fissions. Our goal was to test whether cell type specific differences of higher order chromatin arrangements are evolutionarily conserved in different primate lineages. Whole genome painting experiments and further detailed analyses of individual chromosomes indicate a gene-density-correlated higher order organization of chromatin in lymphoblastoid cell nuclei of all studied primate species, despite evolutionary chromosome reshuffling. In contrast, in primate fibroblast nuclei evolutionary translocations, fissions and fusions resulted in positional shifts of orthologous chromosome segments, thus arguing against a functional role of chromosome size-dependent spatial chromatin arrangements and for geometrical constraints in flat-ellipsoidal fibroblast nuclei. Notably, in both cell types, regions of rearranged chromosomes with distinct differences in gene density showed polarized arrangements with the more gene-dense segment oriented towards the nuclear interior. Our results indicate that nonrandom breakage and rejoining of preferentially gene-dense chromosomes or chromosome segments may have occurred during evolution.

We demonstrate that the nuclear topological arrangement of chromosome territories (CTs) has been conserved during primate evolution over a period of about 30 million years. Recent evidence shows that the positioning of chromatin in human lymphocyte nuclei is correlated with gene density. For example, human chromosome 19 territories, which contain mainly gene-dense and early replicating chromatin, are located toward the nuclear center, whereas chromosome 18 territories, which consist mainly of gene-poor and later replicating chromatin, is located close to the nuclear border. In this study, we subjected seven different primate species to comparative analysis of the radial distribution pattern of human chromosome 18- and 19-homologous chromatin by three-dimensional fluorescence in situ hybridization. Our data demonstrate that gene-density-correlated radial chromatin arrangements were conserved during higher-primate genome evolution, irrespective of the major karyotypic rearrangements that occurred in different phylogenetic lineages. The evolutionarily conserved positioning of homologous chromosomes or chromosome segments in related species supports evidence for a functionally relevant higher-order chromatin arrangement that is correlated with gene-density.

Background The New World monkey (Platyrrhini) subfamily Pitheciinae is represented by the genera Pithecia , Chiropotes and Cacajao . In this work we studied the karyotypes of Pithecia irrorata (2n = 48) and Cacajao calvus rubicundus (2n = 45 in males and 2n = 46 in females) by G- and C-banding, NOR staining and chromosome painting using human and Saguinus oedipus whole chromosome probes. The karyotypes of both species were compared with each other and with Chiropotes utahicki (2n = 54) from the literature. Results Our results show that members of the Pitheciinae have conserved several chromosome forms found in the inferred ancestral Platyrrhini karyotype (associations of human homologous segments 3a/21, 5/7a, 2b/16b, 8a/18, 14/15a and 10a/16a). Further, the monophyly of this subfamily is supported by three chromosomal synapomorphies (2a/10b, an acrocentric 15/14 and an acrocentric human 19 homolog). In addition, each species presents several autapomorphies. From this data set we established a chromosomal phylogeny of Pitheciinae, resulting in a single most parsimonious tree. Conclusions In our chromosomal phylogeny, the genus Pithecia occurred in a more basal position close to the inferred ancestor of Platyrrhini, while C. c. rubicundus and C. utahicki are closely related and are linked by exclusive synapomorphies.

Recently it has been shown that the gene-density correlated radial distribution of human 18 and 19 homologous chromosome territories (CTs) is conserved in higher primates in spite of chromosomal rearrangements that occurred during evolution. However, these observations were limited to apes and New World monkey species. In order to provide further evidence for the evolutionary conservation of gene-density-correlated CT arrangements, we extended our previous study to Old World monkeys. They comprise the remaining species group to be analyzed in order to obtain a comprehensive overview of the nuclear topology of human 18 and 19 homologous CTs in higher primates. In the present study we investigated four lymphoblastoid cell lines from three species of Old World monkeys by three-dimensional fluorescence in situ hybridization (3D-FISH): two individuals of Japanese macaque (Macaca fuscata), crab-eating macaque (Macaca fascicularis), and an interspecies hybrid individual between African green monkey (Cercopithecus aethiops) and Patas monkey (Erythrocebus patas). Our data demonstrate that gene-poor human 18 homologous CTs are located preferentially close to the nuclear periphery, whereas gene-dense human 19 homologous CTs are oriented towards the nuclear center in all cell lines analyzed. The gene-density-correlated positioning of human 18 and 19 homologous CTs is evolutionarily conserved throughout all major higher primate lineages, despite chromosomal inversions, fusions, fissions or reciprocal translocations that occurred in the course of evolution in these species. This remarkable preservation of a gene-density-correlated chromatin arrangement gives further support for a functionally relevant higher-order chromatin architecture.   

We performed multidirectional chromosome painting in a comparative cytogenetic study of the three howler monkey species Alouatta fusca, A. caraya and A. seniculus macconnelli (Atelinae, Platyrrhini) in order to reconstruct phylogenetic relationships within this genus. Comparative genome maps between these species were established by multicolor fluorescence in-situ hybridization (FISH) employing human, Saguinus oedipus and Lagothrix lagothricha chromosome-specific probes. The three species included in this study and previously analyzed howler monkey species were subjected to a phylogenetic analysis on the basis of a data matrix comprised of 98 discrete molecular cytogenetic characters. The results revealed that howler monkeys represent the genus with the most extensive karyotype diversity within Platyrrhini so far analyzed with high levels of intraspecific chromosomal variability. Two different multiple sex chromosome systems were identified. The phylogenetic analysis indicated that Alouatta is a monophyletic clade which can be derived from a proposed ancestral Atelinae karyotype of 2n = 62 chromosomes by a chromosome fusion, a fission, a Y-autosomal translocation and a pericentric inversion. Following these suggestions, the genus Alouatta can be divided into two distinct species groups: the first includes A. caraya and A. belzebul, the second A. s. macconnelli, A. sara, A. s. arctoidea and A. fusca.

We describe a FISH protocol that allows rehybridization of complex DNA probes up to four times to the same specimen. This strategy, which we termed ReFISH, opens a wide range of new applications to conventional band pass filter epifluorescence microscopy. These include M-FISH karyotyping and cross-species color banding that emulate multiplex probe sets labeled with up to 12 fluorochromes in sequential hybridizations to the same specimen. We designed a human 24-color karyotyping probe set in combination with a 29-color cross-species color banding probe set using gibbon painting probes. Applying the ReFISH principle, 53 painting probes on individual metaphases were discriminated. This allowed simultaneous screening for inter- and intrachromosomal rearrangements on normal human diploid cells, a HeLa derived cell line, and highly rearranged gibbon chromosomes. Furthermore, the present ReFISH experiments successfully combine 24-color FISH with laser scanning confocal microscopy to study the 3D organization of all 46 human chromosome territories in individual interphase cell nuclei.[PUBLICATION ABSTRACT]

We describe a FISH protocol that allows rehybridization of complex DNA probes up to four times to the same specimen. This strategy, which we termed ReFISH, opens a wide range of new applications to conventional band pass filter epifluorescence microscopy. These include M-FISH karyotyping and cross-species color banding that emulate multiplex probe sets labeled with up to 12 fluorochromes in sequential hybridizations to the same specimen. We designed a human 24-color karyotyping probe set in combination with a 29-color cross-species color banding probe set using gibbon painting probes. Applying the ReFISH principle, 53 painting probes on individual metaphases were discriminated. This allowed simultaneous screening for inter- and intrachromosomal rearrangements on normal human diploid cells, a HeLa derived cell line, and highly rearranged gibbon chromosomes. Furthermore, the present ReFISH experiments successfully combine 24-color FISH with laser scanning confocal microscopy to study the 3D organization of all 46 human chromosome territories in individual interphase cell nuclei.

The New World monkey (Platyrrhini) subfamily Pitheciinae is represented by the genera Pithecia, Chiropotes and Cacajao. In this work we studied the karyotypes of Pithecia irrorata (2n = 48) and Cacajao calvus rubicundus (2n = 45 in males and 2n = 46 in females) by G- and C-banding, NOR staining and chromosome painting using human and Saguinus oedipus whole chromosome probes. The karyotypes of both species were compared with each other and with Chiropotes utahicki (2n = 54) from the literature. Our results show that members of the Pitheciinae have conserved several chromosome forms found in the inferred ancestral Platyrrhini karyotype (associations of human homologous segments 3a/21, 5/7a, 2b/16b, 8a/18, 14/15a and 10a/16a). Further, the monophyly of this subfamily is supported by three chromosomal synapomorphies (2a/10b, an acrocentric 15/14 and an acrocentric human 19 homolog). In addition, each species presents several autapomorphies. From this data set we established a chromosomal phylogeny of Pitheciinae, resulting in a single most parsimonious tree. In our chromosomal phylogeny, the genus Pithecia occurred in a more basal position close to the inferred ancestor of Platyrrhini, while C. c. rubicundus and C. utahicki are closely related and are linked by exclusive synapomorphies.

The New World monkey (Platyrrhini) subfamily Pitheciinae is represented by the genera Pithecia, Chiropotes and Cacajao. In this work we studied the karyotypes of Pithecia irrorata (2n = 48) and Cacajao calvus rubicundus (2n = 45 in males and 2n = 46 in females) by G- and C-banding, NOR staining and chromosome painting using human and Saguinus oedipus whole chromosome probes. The karyotypes of both species were compared with each other and with Chiropotes utahicki (2n = 54) from the literature. Our results show that members of the Pitheciinae have conserved several chromosome forms found in the inferred ancestral Platyrrhini karyotype (associations of human homologous segments 3a/21, 5/7a, 2b/16b, 8a/18, 14/15a and 10a/16a). Further, the monophyly of this subfamily is supported by three chromosomal synapomorphies (2a/10b, an acrocentric 15/14 and an acrocentric human 19 homolog). In addition, each species presents several autapomorphies. From this data set we established a chromosomal phylogeny of Pitheciinae, resulting in a single most parsimonious tree. In our chromosomal phylogeny, the genus Pithecia occurred in a more basal position close to the inferred ancestor of Platyrrhini, while C. c. rubicundus and C. utahicki are closely related and are linked by exclusive synapomorphies.