Chromosomal instability is a hallmark of cancer and correlates with the presence of extra centrosomes, which originate from centriole overduplication. Overduplicated centrioles lead to the formation of centriole rosettes, which mature into supernumerary centrosomes in the subsequent cell cycle. While extra centrosomes promote chromosome missegregation by clustering into pseudo-bipolar spindles, the contribution of centriole rosettes to chromosome missegregation is unknown. We used multi-modal imaging of cells with conditional centriole overduplication to show that mitotic rosettes in bipolar spindles frequently harbor unequal centriole numbers, leading to biased chromosome capture that favors binding to the prominent pole. This results in chromosome missegregation and aneuploidy. Rosette mitoses lead to viable offspring and significantly contribute to progeny production. We further show that centrosome abnormalities in primary human malignancies frequently consist of centriole rosettes. As asymmetric centriole rosettes generate mitotic errors that can be propagated, rosette mitoses are sufficient to cause chromosome missegregation in cancer.

Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes, and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis, and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy because cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5.

Centrosomes, the main microtubule-organizing centers in most animal cells, are of crucial importance for the assembly of a bipolar mitotic spindle and subsequent faithful segregation of chromosomes into two daughter cells. Centrosome abnormalities can be found in virtually all cancer types and have been linked to chromosomal instability (CIN) and tumorigenesis. Although our knowledge on centrosome structure, replication, and amplification has greatly increased within recent years, still only very little is known on nature, causes, and consequences of centrosome aberrations in primary tumor tissues. In this review, we summarize our current insights into the mechanistic link between centrosome aberrations, aneuploidy, CIN and tumorigenesis. Mechanisms of induction and cellular consequences of aneuploidy, tetraploidization and CIN, as well as origin and effects of supernumerary centrosomes will be discussed. In addition, animal models for both CIN and centrosome amplification will be outlined. Finally, we describe approaches to exploit centrosome amplification, aneuploidy and CIN for novel and specific anticancer treatment strategies based on the modulation of chromosome missegregation rates.

Somatic rearrangements resulting in genomic structural variation drive malignant phenotypes by altering the expression or function of cancer genes. Pan-cancer studies have revealed that structural variants (SVs) are the predominant class of driver mutation in most cancer types, but because they are difficult to discover, they remain understudied when compared with point mutations. This review provides an overview of the current knowledge of somatic SVs, discussing their primary roles, prevalence in different contexts, and mutational mechanisms. SVs arise throughout the life history of cancer, and 55% of driver mutations uncovered by the Pan-Cancer Analysis of Whole Genomes project represent SVs. Leveraging the convergence of cell biology and genomics, we propose a mechanistic classification of somatic SVs, from simple to highly complex DNA rearrangement classes. The actions of DNA repair and DNA replication processes together with mitotic errors result in a rich spectrum of SV formation processes, with cascading effects mediating extensive structural diversity after an initiating DNA lesion has formed. Thanks to new sequencing technologies, including the sequencing of single-cell genomes, open questions about the molecular triggers and the biomolecules involved in SV formation as well as their mutational rates can now be addressed. Expected final online publication date for the Annual Review of Genomics and Human Genetics, Volume 23 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Summary We report a droplet microfluidic method to target and sort individual cells directly from complex microbiome samples and to prepare these cells for bulk whole-genome sequencing without cultivation. We characterize this approach by recovering bacteria spiked into human stool samples at a ratio as low as 1:250 and by successfully enriching endogenous Bacteroides vulgatus to the level required for de novo assembly of high-quality genomes. Although microbiome strains are increasingly demanded for biomedical applications, a vast majority of species and strains are uncultivated and without reference genomes. We address this shortcoming by encapsulating complex microbiome samples directly into microfluidic droplets and amplifying a target-specific genomic fragment using a custom molecular TaqMan probe. We separate those positive droplets by droplet sorting, selectively enriching single target strain cells. Finally, we present a protocol to purify the genomic DNA while specifically removing amplicons and cell debris for high-quality genome sequencing.

Chromosomal instability is one of the hallmarks of cancer and caused by chromosome missegregation during mitosis, a process frequently associated with micronucleus formation. Micronuclei are formed when chromosomes fail to join a daughter nucleus during cell division and are surrounded by their own nuclear membrane. Although it has been commonly assumed that the gain or loss of specific chromosomes is random during compromised cell division, recent data suggest that the size of chromosomes can impact on chromosome segregation fidelity. To test whether chromosome missegregation rates scale with chromosome size in primary human cancer cells, we assessed chromosome sequestration into micronuclei in patient‐derived primary NCH149 glioblastoma cells, which display high‐level numerical chromosome instability (CIN), pronounced spontaneous micronucleus formation but virtually no structural CIN. The cells were analyzed by interphase fluorescence in situ hybridization using chromosome‐specific painting probes for all chromosomes. Overall, 33% of early passage NCH149 cells harbored micronuclei. Entrapment within a micronucleus clearly correlated with chromosome size with larger chromosomes being significantly more frequently missegregated into micronuclei than smaller chromosomes in primary glioblastoma cells. These findings extend the concept that chromosome size determines segregation fidelity by implying that size‐specific micronucleus entrapment occurs in primary human cancer cells as well.

Introduction Genomic instability is the basic prerequisite for a Darwinian-type evolution of neoplasia and as such represents a fundamental hallmark of cancer. Centrosomal aberrations have been identified as potent drivers of genomic instability (Cosenza et al., Cell Reports 2017; Krämer et al., Leukemia 2003). The current standard to investigate centrosomal aberrations in cancer patients is immunofluorescence (IF) staining. Although this method is fast and easily scalable, its diagnostic significance is controversially discussed. Moreover, ultrastructural analysis of centrosomes in cancer patients is required to gain a mechanistical understanding of the relationship between genomic instability and centrosomal aberrations. To address this, we combined semi-automated analysis of immunofluorescence (IF) images with high-throughput electron tomography (ET) of different cell lines and subentities of primary plasma cell neoplasia, which serve as surrogate for clonal evolution. Methods CD138+ plasma cells were isolated from bone marrow aspirates of consenting patients with plasma cell neoplasia. Each sample was split to be subsequently processed for IF and ET. The IF workflow included (1) chemical fixation, (2) staining for nuclei, cells, centrin and pericentrin, (3) semi-automated acquisition of >1000 cells, (4) semi-automated analysis of IF data using the software Konstanz Information Miner (KNIME) (Berthold et al., GfKL 2007). The ET workflow included (1) chemical fixation (2) agarose embedding, (3) dehydration and epoxy resin embedding, (4) serial sectioning at 200 nm, (5) semi-automated screening for centrioles with transmission electron microscopy (TEM) (Schorb et al., Nature Methods 2019), (6) semi-automated acquisition of previously identified centriole regions with serial section ET. Results So far, four patients with relapsed refractory myeloma as well as two cell lines (U2OS-PLK4, RPMI.8226) have been screened with TEM. No centrosomal amplification was apparent by IF in any of these patients. Within 5598 cells, 205 centrosomes have been detected. A total of 659 electron tomograms were performed on 141 regions of interest that were distributed on average over five sections. One patient with highly refractory multiple myeloma (resistance to eight prior therapies) showed over-elongated and partially fragmented centrioles (Figure), similar to recently reported findings in tumor cell lines (Marteil et al., Nature Communications 2018). Six out of 10 mother centrioles in this patient were longer than 500 nm, which is supposed to be the physiological length. The dimensions (mean [range]) of mother (decorated with appendages) and daughter centrioles in this patient were: length 919 nm [406 nm - 2620 nm] and 422 nm [367 nm - 476 nm]; diameter 221 nm [99 nm - 470 nm] and 236 nm [178 nm - 450 nm]. Moreover, the mother centrioles showed multiple sets of appendages (mean [range]: 5.9 [2 - 13]), while one set of appendages would be physiological. This is an ongoing study and additional results are expected by the date of presentation. Conclusions We present a semi-automated methodological setup that combines high-throughput IF and cutting-edge ET to study centrosomal aberrations. To our knowledge, this is the first study that systematically analyzes the centrosomal phenotype of cancer patients at the ultrastructural level. Our preliminary IF results suggest that supernumerary centrosomes in plasma cell neoplasia might be less common than previously reported. Moreover, we for the first time describe and characterize over-elongated centrioles in myeloma patients, reminiscent of previous findings in tumor cell lines. With increasing numbers of patients, we will be also able to correlate results from IF and ET to address the current uncertainty with respect to IF screens for centrosomal aberrations. Better insight into centrosomal aberrations will likely increase our understanding on karyotype evolution in plasma cell neoplasia and possibly facilitate the development of novel targeted therapies. Figure Disclosures Goldschmidt: John-Hopkins University: Research Funding; John-Hopkins University: Research Funding; MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Molecular Partners: Research Funding; Janssen: Consultancy, Research Funding; Mundipharma: Research Funding; Chugai: Honoraria, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Schönland:Medac: Other: Travel Grant; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Krämer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding.

Summary: Single-cell DNA template strand sequencing (Strand-seq) allows a range of various genomic analysis including chromosome length haplotype phasing and structural variation (SV) calling in individual cells. Here, we present MosaiCatcher v2, a standardised workflow and reference framework for single-cell SV detection using Strand-seq. This framework introduces a range of functionalities, including: an automated upstream Quality Control (QC) and assembly sub-workflow that relies on multiple genome assemblies and incorporates a multistep normalisation module, integration of the scNOVA SV functional characterization and of the ArbiGent SV genotyping modules, platform portability, as well as a user-friendly and shareable web report. These new features of MosaiCatcher v2 enables reproducible computational processing of Strand-seq data, which are increasingly used in human genetics and single cell genomics, towards production environments. Availability and Implementation: Mosaicatcher v2 is a standardised workflow, implemented using the Snakemake workflow management system. The pipeline is available on GitHub: https://github.com/friendsofstrandseq/mosaicatcher-pipeline/ and on the snakemake-workflow-catalog: https://snakemake.github.io/snakemake-workflow-catalog/?usage=friendsofstrandseq/mosaicatcher-pipeline. Contact: jan.korbel@embl.de