A fluorescence in situ hybridization-based screen allows rapid detection of adverse cytogenetic alterations in patients with acute myeloid leukemia.

In adult acute myeloid leukemia (AML), the karyotype of the leukemic cell is among the strongest prognostic factors. The Medical Research Council (MRC) and the European LeukemiaNet (ELN) classifications distinguish between favorable, intermediate and adverse cytogenetic risk patients who differ in their treatment response and overall survival. Conventional cytogenetic analyses are a mandatory component of AML diagnostics but they are time-consuming; therefore, therapeutic decisions in elderly patients are often delayed. We investigated whether a screening approach using a panel of seven fluorescence in situ hybridization (FISH) probes would allow rapid identification of adverse chromosomal changes. In a cohort of 334 AML patients, our targeted FISH screening approach identified 80% of adverse risk AML patients with a specificity of 99%. Incorporating FISH screening into diagnostic workup has the potential to accelerate risk stratification and treatment selection, particularly in older patients. This approach may allow therapeutic decisions more quickly, which benefits both patients and physicians and might save costs.

Distinct nuclear orientation patterns for mouse chromosome 11 in normal B lymphocytes.

BACKGROUND: Characterizing the nuclear orientation of chromosomes in the three-dimensional (3D) nucleus by multicolor banding (mBANDing) is a new approach towards understanding nuclear organization of chromosome territories. An mBANDing paint is composed of multiple overlapping subchromosomal probes that represent different regions of a single chromosome. In this study, we used it for the analysis of chromosome orientation in 3D interphase nuclei. We determined whether the nuclear orientation of the two chromosome 11 homologs was random or preferential, and if it was conserved between diploid mouse Pre B lymphocytes of BALB/c origin and primary B lymphocytes of congenic [T38HxBALB/c]N wild-type mice. The chromosome orientation was assessed visually and through a semi-automated quantitative analysis of the radial and angular orientation patterns observed in both B cell types. RESULTS: Our data indicate that there are different preferential patterns of chromosome 11 orientation, which are not significantly different between both mouse cell types (p > 0.05). In the most common case for both cell types, both copies of chromosome 11 were oriented in parallel with the nuclear border. The second most common pattern in both types of B lymphocytes was with one homolog of chromosome 11 positioned with its telomeric end towards the nuclear center and with its centromeric end towards the periphery, while the other chromosome 11 was found parallel with the nuclear border. In addition to these two most common orientations present in approximately 50% of nuclei from each cell type, other orientations were observed at lower frequencies. CONCLUSIONS: We conclude that there are probabilistic, non-random orientation patterns for mouse chromosome 11 in the mouse B lymphocytes we investigated (p < 0.0001).

Double Drop-Off Droplet Digital PCR: A Novel, Versatile Tool for Mutation Screening and Residual Disease Monitoring in Acute Myeloid Leukemia Using Cellular or Cell-Free DNA.

In acute myeloid leukemia (AML), somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed. Current techniques for mutation screening and quantification, including next-generation sequencing and quantitative PCR, each have weaknesses that leave a need for novel diagnostic tools. We established double drop-off digital droplet PCR (DDO-ddPCR) assays for gene mutations in NPM1, IDH2, and NRAS, which can detect and quantify diverse alterations at two nearby hotspot regions present in these genes. These assays can be used for mutation screening as well as quantification and sequential monitoring. The assays were validated against next-generation sequencing and existing ddPCR assays and achieved high concordance with an overall sensitivity comparable to conventional digital PCR. In addition, the feasibility of detecting and monitoring genetic alterations in peripheral blood cell-free DNA (cfDNA) of patients with AML by DDO-ddPCR was studied. cfDNA analysis was found to have similar sensitivity compared to quantitative PCR-based analysis of peripheral blood. Finally, the cfDNA-based digital PCR in several clinical scenarios was found to be useful in long-term monitoring of target-specific therapy, early response assessment during induction chemotherapy, and identification of mutations in patients with extramedullary disease. Thus, DDO-ddPCR-based cfDNA analysis may complement existing genetic tools for diagnosis and disease monitoring in AML.

Chromosome phylogeny of the subfamily Pitheciinae (Platyrrhini, Primates) by classic cytogenetics and chromosome painting.

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.

Validation and refinement of the revised 2017 European LeukemiaNet genetic risk stratification of acute myeloid leukemia.

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.

Evolutionary genomic remodelling of the human 4q subtelomere (4q35.2).

BACKGROUND: In order to obtain insights into the functionality of the human 4q35.2 domain harbouring the facioscapulohumeral muscular dystrophy (FSHD) locus, we investigated in African apes genomic and chromatin organisations, and the nuclear topology of orthologous regions. RESULTS: A basic block consisting of short D4Z4 arrays (10-15 repeats), 4q35.2 specific sequences, and approximately 35 kb of interspersed repeats from different LINE subfamilies was repeated at least twice in the gorilla 4qter. This genomic organisation has undergone evolutionary remodelling, leading to the single representation of both the D4Z4 array and LINE block in chimpanzee, and the loss of the LINE block in humans. The genomic remodelling has had an impact on 4qter chromatin organisation, but not its interphase nuclear topology. In comparison with humans, African apes show very low or undetectable levels of FRG1 and FRG2 histone 4 acetylation and gene transcription, although histone deacetylase inhibition restores gene transcription to levels comparable with those of human cells, thus indicating that the 4qter region is capable of acquiring a more open chromatin structure. Conversely, as in humans, the 4qter region in African apes has a very peripheral nuclear localisation. CONCLUSION: The 4q subtelomere has undergone substantial genomic changes during evolution that have had an impact on chromatin condensation and the region's transcriptional regulation. Consequently, the 4qter genes in African apes and humans seem to be subjected to a different strategy of regulation in which LINE and D4Z4 sequences may play a pivotal role. However, the effect of peripheral nuclear anchoring of 4qter on these regulation mechanisms is still unclear. The observed differences in the regulation of 4qter gene expression between African apes and humans suggest that the human 4q35.2 locus has acquired a novel functional relevance.

Increased chromosome 16 disomy rates in human spermatozoa and recurrent spontaneous abortions.

OBJECTIVE: To investigate if unexplained recurrent spontaneous abortions (RSA) are associated with increased rates of aneuploidy in spermatozoa of RSA partners ("RSA-men"). DESIGN: Case-control study. SETTING: Academic research center. PATIENT(S): Patients enrolled at the Hormone and Fertility Center and controls at the Department of Urology (LMU-Munich). INTERVENTION(S): Sperm samples of 11 partners of unexplained RSA cases evaluated for elevated diploidy and disomy levels of chromosomes 1-22, X, and Y by multicolor sperm fluorescence in situ hybridization (FISH). MAIN OUTCOME MEASURE(S): Aneuploidy rates obtained in RSA-men compared with controls from the literature and internally; an increase of the aneuploidy rate was considered statistically significant, when it differed ≥ 2 standard deviations from the mean baseline level in controls. RESULT(S): Our sperm FISH data on RSA men showed increased disomy rates for at least three chromosomes in more than 60% of patients but no statistically significant increase of the overall mean sperm disomy or diploidy rate. In particular, meiotic errors involving chromosome 16 contributed to increased sperm disomy in more than 60% of our patients. CONCLUSION(S): These data suggest that among paternal meiotic errors nondisjunction of chromosome 16 might have similar relative influence on fetal aneuploidy compared with maternal chromosome 16 disomy.

Replication-timing-correlated spatial chromatin arrangements in cancer and in primate interphase nuclei.

Using published high-resolution data on S-phase replication timing, we determined the three-dimensional (3D) nuclear arrangement of 33 very-early-replicating and 31 very-late-replicating loci. We analyzed diploid human, non-human primate and rearranged tumor cells by 3D fluorescence in situ hybridization with the aim of investigating the impact of chromosomal structural changes on the nuclear organization of these loci. Overall, their topology was found to be largely conserved between cell types, species and in tumor cells. Early-replicating loci were localized in the nuclear interior, whereas late-replicating loci showed a broader distribution with a higher preference for the periphery than for late-BrdU-incorporation foci. However, differences in the spatial arrangement of early and late loci of chromosome 2, as compared with those from chromosome 5, 7 and 17, argue against replication timing as a major driving force for the 3D radial genome organization in human lymphoblastoid cell nuclei. Instead, genomic properties, and local gene density in particular, were identified as the decisive parameters. Further detailed comparisons of chromosome 7 loci in primate and tumor cells suggest that the inversions analyzed influence nuclear topology to a greater extent than the translocations, thus pointing to geometrical constraints in the 3D conformation of a chromosome territory.

Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.

Fluorescence in situ hybridization (FISH) of specific DNA probes has become a widely used technique mostly for chromosome analysis and for studies of the chromosomal location of specific DNA segments in metaphase preparations as well as in interphase nuclei. FISH on 3D-preserved nuclei (3D-FISH) in combination with 3D-microscopy and image reconstruction is an efficient tool to analyze the spatial arrangement of targeted DNA sequences in the nucleus. Recent developments of a "new generation" of confocal microscopes that allow the distinct visualization of at least five different fluorochromes within one experiment opened the way for multicolor 3D-FISH experiments. Thus, numerous differently labeled nuclear targets can be delineated simultaneously and their spatial interrelationships can be analyzed on the level of individual nuclei.In this chapter, we provide protocols for the preparation of complex DNA-probe sets suitable for 3D-FISH with up to six different fluorochromes, for 3D-FISH on cultured mammalian cells (growing in suspension or adherently) as well as on tissue sections, and for 3D immuno-FISH.In comparison with FISH on metaphase chromosomes and conventional interphase cytogenetics, FISH on 3D-preserved nuclei requires special demands with regard to probe quality, fixation, and pretreatment steps of cells in order to achieve the two goals, namely the best possible preservation of the nuclear structure and at the same time an efficient probe accessibility.

Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes.

INTRODUCTIONDNA probes for fluorescence in situ hybridization (FISH) can be generated and labeled by various methods. This protocol describes the conjugation of dUTPs with haptens or fluorochromes, as well as the generation and labeling of DNA probes using those modified dUTPs. Sources of probe DNA include genomic DNA, DNA from flow-sorted chromosomes, bacterial artificial chromosomes (BACs), and cosmids. DNA amplification and labeling procedures involving degenerate oligonucleotide-primed PCR (DOP-PCR) and multiple displacement amplification (MDA) are provided. Advice is given for setting up complex probe pools, such as those containing large pools of BAC probes. Also included is a method for probe precipitation and preparation of a hybridization mix ready to be used for 3D fluorescence in situ hybridization (FISH) experiments.

Evolutionarily conserved, cell type and species-specific higher order chromatin arrangements in interphase nuclei of primates.

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.

Towards unlimited colors for fluorescence in-situ hybridization (FISH).

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 evolutionary history of human chromosome 7.

We report on a comparative molecular cytogenetic and in silico study on evolutionary changes in human chromosome 7 homologs in all major primate lineages. The ancestral mammalian homologs comprise two chromosomes (7a and 7b/16p) and are conserved in carnivores. The subchromosomal organization of the ancestral primate segment 7a shared by a lemur and higher Old World monkeys is the result of a paracentric inversion. The ancestral higher primate chromosome form was then derived by a fission of 7b/16p, followed by a centric fusion of 7a/7b as observed in the orangutan. In hominoids two further inversions with four distinct breakpoints were described in detail: the pericentric inversion in the human/African ape ancestor and the paracentric inversion in the common ancestor of human and chimpanzee. FISH analysis employing BAC probes confined the 7p22.1 breakpoint of the pericentric inversion to 6.8 Mb on the human reference sequence map and the 7q22.1 breakpoint to 97.1 Mb. For the paracentric inversion the breakpoints were found in 7q11.23 between 76.1 and 76.3 Mb and in 7q22.1 at 101.9 Mb. All four breakpoints were flanked by large segmental duplications. Hybridization patterns of breakpoint-flanking BACs and the distribution of duplicons suggest their presence before the origin of both inversions. We propose a scenario by which segmental duplications may have been the cause rather than the result of these chromosome rearrangements.

The phylogeny of howler monkeys (Alouatta, Platyrrhini): reconstruction by multicolor cross-species chromosome painting.

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.

Evolutionary conservation of chromosome territory arrangements in cell nuclei from higher primates.

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.