Chromoanagenesis is a catastrophic genomic phenomenon involving sudden, extensive rearrangements within one or a few cell cycles. In osteosarcoma, the most prevalent malignant bone tumor in children and adolescents, these events dramatically alter the genomic landscape, frequently disrupting key tumor suppressor genes like TP53 and RB1, amplifying oncogene expression, and propelling tumor progression and evolution. This review elucidates how key chromoanagenic mechanisms, such as chromothripsis and chromoanasynthesis, arise from replication stress and impaired DNA repair pathways, ultimately contributing to genomic instability in osteosarcoma. Chromothripsis features prominently in osteosarcoma, occurring in up to 62% of tumor regions and driving intratumoral heterogeneity through persistent genomic crises. Next-generation sequencing, optical genome mapping, and emerging technologies like single-cell sequencing empower researchers to detect and characterize these complex structural variants, demonstrating how a single catastrophic event can profoundly influence osteosarcoma progression over time. While targeted therapies for osteosarcoma have proven elusive, innovative strategies harnessing comprehensive genomic profiling and patient-derived preclinical models hold promise for uncovering tumor-specific vulnerabilities tied to chromoanagenesis. Ultimately, unraveling how these rapid, large-scale rearrangements fuel osteosarcoma's aggressive nature will not only refine disease classification and prognosis but also pave the way for novel therapeutic approaches to enhance patient outcomes.

5q14.3 microdeletion syndrome (MIM#613443) is a neurodevelopmental disorder (NDD) involving copy number loss of multiple genes including Myocyte enhancer factor 2C (MEF2C) gene in the q14.3 region of chromosome 5. Chromosomes 5 and 15 chromothripsis involving 5q14.3 was previously reported in one individual with developmental epileptic encephalopathy (DEE). A complex chromothripsis between chromosomes 3, 5, 7, 9, and 18 that involved 5q14.3 was also reported in a pregnancy complicated by brain and kidney anomalies on fetal ultrasound. Here, we report chromothripsis of chromosomes 3q and 5q involving 5q14.3 in a three-year-old female with Lennox-Gastaut syndrome. The chromosomes 3q and 5q chromothripsis was found by trio genome sequencing (GS) and confirmed by fluorescent in situ hybridization (FISH). Notable clinical findings include medically refractory seizures, global developmental delay, increased fluid-attenuated inversion recovery (FLAIR) signal in the left inferior temporal gyrus, and dysmorphic features. Chromothripsis of chromosomes 3 and 5 was previously recognized in renal cell carcinomas. To the best of our knowledge, this is the first reported case of chromosomes 3q and 5q chromothripsis leading to a developmental epileptic encephalopathy (DEE) due to disruption of 5q14.3. These findings expand chromosomes 3 and 5 chromothripsis as a genomic mechanism underlying 5q14.3 microdeletion syndrome.

A histological evaluation remains the cornerstone of diagnosing highly malignant osteosarcoma, having demonstrated its efficacy and reliability over several decades. However, despite these advancements, misdiagnoses with severe consequences, including inadequate surgical procedures, continue to occur. Consequently, there is a pressing need to further enhance diagnostic security. Adjunct immunohistochemical approaches have demonstrated significant effectiveness in regard to cancer diagnostics, generally. However, their utility for identifying highly malignant osteosarcoma is limited. Molecular genetic findings have significantly improved the diagnosis of Ewing's sarcoma by identifying specific translocations and have been used to detect specific IDH gene mutations in chondrosarcoma. Nevertheless, molecular genetic alterations in highly malignant osteosarcoma exhibit a high degree of complexity, thereby limiting their diagnostic utility. Given that only 1-2% of the human genome comprises protein-coding sequences, the growing number of non-coding regulatory RNAs, which are increasingly being elucidated, has garnered substantial attention in the field of clinical cancer diagnostics. Over the past several years, patterns of altered non-coding RNA expression have been identified that facilitate the distinction between benign and malignant tumors in various organs. In the field of bone tumors, the experience of this approach has been limited thus far. The divergent expression of microRNAs has demonstrated utility for differentiating osteosarcoma from osteoblastoma and discriminating between osteosarcoma and giant-cell tumors of bone and fibrous dysplasia. However, the application of non-coding RNA expression patterns for the differential diagnosis of osteosarcoma is still in its preliminary stages. This review provides an overview of the current status of non-coding RNAs in osteosarcoma diagnostics, in conjunction with a histological evaluation. The potential of this approach is discussed comprehensively.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a short survival time after standard therapy administration including radiotherapy (RT) associated with temozolomide (TMZ). Here, we investigated the effects of radiochemotherapy in association with metformin (MET), a drug targeting cell metabolism on a syngeneic GBM mouse model using Positron Emission Tomography imaging with [18F]FLT and [18F]VC701 and single-cell RNA-sequencing analysis. The addition of drugs to RT significantly increased survival and [18F]FLT showed an early predictive response of combined therapy. We identified the presence of heterogeneous tumor populations with different treatment sensitivity and a complex immune evasive microenvironment. Tumor cells surviving to treatments showed immune response, among the main differentially modulated biological functions and a potential role of long non-coding RNAs (lncRNAs) in treatment resistance. Association with TMZ or TMZ plus MET reduced the pro-tumor phenotype of immune reaction acting more on myeloid cells the first and on lymphocytes the latter. Off note, MET add-on counteracted the immune-evasive phenotype particularly of T cells suggesting a potential role of MET also in adopted immunity.

Micronuclei exhibit defective proteomes rendering their chromatin vulnerable to fragmentation. This fragmentation process, known as chromothripsis, promotes tumorigenesis by catalysing the activation of oncogenes and the silencing of tumor suppressors. With this role in mind, micronuclei serve as promising targets for therapeutic intervention. This review will explore recent discoveries regarding how micronuclei form, their function in catalysing chromothripsis and how chromothripsis provides a selective advantage for cancer cells.

Osteosarcoma is one of the most prevalent malignant bone tumors, and despite advances in treatment, significant improvements in survival rates for osteosarcoma patients remain elusive. There is an urgent need for developing novel molecules for the targeted treatment of osteosarcoma. NAD(P)H:quinone oxidoreductase 1 (NQO1) is highly overexpressed in osteosarcoma. Here, we evaluated a series of in-house NQO1-targeting compounds, including NQO1 substrates and β-lap prodrugs, through phenotypic screening using NQO1-positive methylnitronitrosoguanidine-induced human osteosarcoma cells (MNNG) and NQO1-negative normal human umbilical vein endothelial cells (HUVEC), aiming to identify novel candidate compounds for osteosarcoma therapy. As a result, compound 21, an NQO1 substrate, was identified as a potent anti-osteosarcoma agent that promotes apoptosis and induces cell cycle arrest in osteosarcoma cells in vitro, while significantly inhibiting tumor growth in vivo. These findings suggest that compound 21 holds promise as a candidate for osteosarcoma treatment. Moreover, NQO1-targeting substrates present a promising pathway for the discovery of novel anti-osteosarcoma agents.

B chromosomes (Bs) exist in addition to the standard (A) chromosomes in a wide range of species. The process underlying their origin is still unclear. We propose pathways of intra- and interspecific origin of B chromosomes based on known mechanisms of chromosome evolution and available knowledge of their sequence composition in different species. We speculate that a mitotic or meiotic segregation error of one or more A chromosomes initiates, via chromoanagenesis, the formation of a proto-B chromosome. In the second step, proto-B chromosomes accumulate A chromosome- and organelle-derived sequences over time, most likely via DNA double-strand break (DSB) mis-repair. Consequently, the original structure of the early stage proto-B chromosomes becomes masked by continuous sequence incorporation. The similarity between A chromosome sequences integrated into B chromosomes and the original sequences on the donor chromosomes decreases over time if there is no selection pressure on these sequences on B chromosomes. However, besides chromoanagenesis, also other mechanisms leading to the formation of B chromosomes might exist.

Chromoplexy is a phenomenon of complex genome rearrangement, occurring during a single cell event and characterized by the formation of chain rearrangements affecting multiple chromosomes. Unlike other genomic rearrangements such as chromothripsis, which involves a single chromosome, chromoplexy affects several chromosomes at once, creating patterns of complex, balanced translocations, and leading to the formation of fusion genes and the simultaneous disruption of several genes. Chromoplexy was first identified in prostate cancers, but it is now observed in various cancers where gene fusions take place. The precise mechanisms behind chromoplexy remain under investigation. The occurrence of these rearrangements follows multiple double-stranded breaks that appear to occur in certain regions or during particular genome configurations (open chromatin, active transcription area), and which lead to an intricate series of inter- and intra-chromosomal translocations and deletions without significant alterations in the number of copies. Although chromoplexy is considered a very early event in oncogenesis, the phenomenon can be repeated and can constitute a mechanism of clonal tumor progression. The occurrence of chromoplexy supports the equilibrium model punctuated by tumor evolution, characterized by periods of relative stability punctuated by sudden and rapid periods of radical genomic changes.

Cancer is fundamentally a disease of the genome, characterized by extensive genomic, transcriptomic, and epigenomic alterations. Most current studies predominantly use short-read sequencing, gene panels, or microarrays to explore these alterations; however, these technologies can systematically miss or misrepresent certain types of alterations, especially structural variants, complex rearrangements, and alterations within repetitive regions. Long-read sequencing is rapidly emerging as a transformative technology for cancer research by providing a comprehensive view across the genome, transcriptome, and epigenome, including the ability to detect alterations that previous technologies have overlooked. In this Perspective, we explore the current applications of long-read sequencing for both germline and somatic cancer analysis. We provide an overview of the computational methodologies tailored to long-read data and highlight key discoveries and resources within cancer genomics that were previously inaccessible with prior technologies. We also address future opportunities and persistent challenges, including the experimental and computational requirements needed to scale to larger sample sizes, the hurdles in sequencing and analyzing complex cancer genomes, and opportunities for leveraging machine learning and artificial intelligence technologies for cancer informatics. We further discuss how the telomere-to-telomere genome and the emerging human pangenome could enhance the resolution of cancer genome analysis, potentially revolutionizing early detection and disease monitoring in patients. Finally, we outline strategies for transitioning long-read sequencing from research applications to routine clinical practice.

The Fibroblast growth factor receptors 3-transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3, F3-T3) oncogenic fusion gene, identified in malignant tumors such as gliomas and bladder cancer, has been particularly noted in recurrent gliomas where it is considered to drive malignant progression, thus presenting itself as a viable therapeutic target. However, the precise mechanism by which F3-T3 facilitates the malignant progression of glioma is not fully understood.

Correction analysis of STAT3 and FGFR3 with major glioma mutation types and pan-cancer analysis was conducted using The Cancer Genome Atlas (TCGA) database. A series of phenotypic experiments, including CCK-8, EdU, colony-formation assay, wound healing assay, and transwell assay were conducted to detect the effects of F3-T3 on proliferation, invasion, and migration of glioma cells. The association between F3-T3 and epithelial-mesenchymal transition (EMT) was investigated through enrichment analysis of the E-MTAB-6037 gene chip database and confirmed by western blot. The underling mechanism were further inferred and validated through RNA sequencing, E-MTAB-6037 gene chip data, and western blot. The relationship between p-STAT3 expression and the WHO grade of glioma was evaluated using immunohistochemistry (IHC) and tissue microarray analysis. Furthermore, the results of vivo experiments and IHC has confirmed the impact of F3-T3 on glioma malignant progression and activation of the STAT3 signaling pathway.

The experimental results from this study indicate that F3-T3 accelerates the epithelial-mesenchymal transition (EMT) process in glioma cells, thereby promoting their proliferation, invasion, and migration capabilities. Mechanistically, it was determined through RNA sequencing that the signal transducer and activator of transcription 3 (STAT3) signaling pathway is crucial for the malignant progression of F3-T3. This finding was further supported through follow-up experiments conducted after STAT3 knockdown. The role of the STAT3 pathway in gliomas was also reinforced through bioinformatic analysis and immunohistochemistry (IHC) on tissue microarrays (TMA). Further in vivo experiments corroborated the role of F3-T3 in enhancing glioma growth and progression.

F3-T3 facilitates the proliferation, invasion, migration and EMT of glioma cells, thereby promoting their malignant progression through STAT3 signaling activation. These findings highlight its potential as a therapeutic target for glioma treatment.

Colorectal carcinoma (CRC) exhibits metastatic organotropism, primarily targeting liver, lung, and rarely the brain. Here, we study chromosomal imbalances (CIs) in cohorts of primary CRCs and metastases. Brain metastases show the highest burden of CIs, including aneuploidies and focal CIs, with enrichment of +12p encoding KRAS. Compared to liver and lung metastases, brain metastases present with increased co-occurrence of KRAS mutation and amplification. CRCs with concurrent KRAS mutation and amplification display significant metabolic reprogramming with upregulation of glycolysis, alongside upregulation of cell cycle pathways, including copy number gains of MDM2 and CDK4. Evolutionary modeling suggests early acquisition of many organotropic CIs enriched in both liver and brain metastases, while brain-enriched CIs preferentially emerge later. Collectively, this study supports a model where cytogenetic events in CRCs favor site-specific metastatic colonization. These site-enriched CI patterns may serve as biomarkers for metastatic potential in precision oncology.

Tandem duplicator phenotype (TDP) consists of distinct genomic rearrangements where tandem duplications are randomly distributed. In this study, we characterized the prevalence and outcomes of TDP in a large series of prospectively sequenced tumors from patients with pancreatic ductal adenocarcinomas (PDAC). Whole-genome sequencing (WGS) was performed in 530 PDAC cases from the PanCuRx Initiative, COMPASS and PanGen/POG trials in Canada. Of 530 cases, 52 were identified as TDP (9.8%; 13 resected, 39 advanced). Etiological subgroups of TDP included BRCA1 (n = 9), CCNE1 (n = 4), and unknown (n = 39). Presence of TDP was not prognostic in resected specimens (p = 0.77) compared with non-HRD and non-TDP cases, described as typicals. In advanced cases, when stratified for only classical subtype cases, platinum therapy was correlated with longer response in non-BRCA1 TDP vs. typicals (p = 0.0036). There was no difference in overall survival between TDP and typicals (p = 0.5).TDP represents a potential novel targetable subgroup for chemotherapy selection in PDAC.

For the detection of somatic structural variation (SV) in cancer genomes, long-read sequencing is advantageous over short-read sequencing with respect to mappability and variant phasing. However, most current long-read SV detection methods are not developed for the analysis of tumor genomes characterized by complex rearrangements and heterogeneity. Here, we present Severus, a breakpoint graph-based algorithm for somatic SV calling from long-read cancer sequencing. Severus works with matching normal samples, supports unbalanced cancer karyotypes, can characterize complex multibreak SV patterns and produces haplotype-specific calls. On a comprehensive multitechnology cell line panel, Severus consistently outperforms other long-read and short-read methods in terms of SV detection F1 score (harmonic mean of the precision and recall). We also illustrate that compared to long-read methods, short-read sequencing systematically misses certain classes of somatic SVs, such as insertions or clustered rearrangements. We apply Severus to several clinical cases of pediatric leukemia/lymphoma, revealing clinically relevant cryptic rearrangements missed by standard genomic panels.

In 1953, Danely Slaughter proposed the concept of field cancerization, or field effect, to explain the development of additional neoplasia of similar type. A recent theory (de Groen, 2022) states that all DNA is exposed to a constant source of damage, resulting in a constant rate of germline and somatic DNA mutations. If the field effect and constant mutation theories are correct and a single somatic mutation causes the transition from non-neoplastic to neoplastic phenotype, then all rates of neoplasia formation can be modeled by exponential equations containing a single variable that determines the chance of phenotype transition. In this perspective, studies from 1953 till 2021 originating from America, Europe, and Asia about head, chest, abdomen, pelvic, and skin neoplasia were reviewed and showed consistent field effects that are modeled by tapering exponential equations containing a single variable defining field effect strength; Pearson and linear correlation coefficients for observed and modeled data range from 0.994 to 1. Thus, existing data are compatible with a constant rate of DNA damage. Organ-specific, tissue-specific, or body-wide mutagenesis conditions determine the rate of neoplasia development and explain the co-occurrence of seemingly unrelated neoplasia at predictable frequencies. Shared risk factors explain increased risk for additional neoplasia in persons with one neoplastic lesion.

Uterine leiomyomas or fibroids are benign tumors of the myometrium that affect approximately 70% of reproductive-age women. Fibroids continue to be the leading cause of hysterectomy, resulting in substantial healthcare costs. Genetic complexity and lack of cellular and molecular understanding of fibroids have posed considerable challenges to developing noninvasive treatment options. Over the years, research efforts have intensified to unravel the genetic and cellular diversities within fibroids to deepen our understanding of their origins and progression. Studies using immunostaining and flow cytometry have revealed cellular heterogeneity within these tumors. A correlation has been observed between genetic mutations in fibroids and their size, which is influenced by cellular composition, proliferation rates, and extracellular matrix accumulation. Fibroids with mediator complex subunit 12 (MED12) mutation are composed of smooth muscle cells and fibroblasts equally. In contrast, the fibroids with high-mobility group AT-hook 2 (HMGA2) translocation are 90% composed of smooth muscle cells. More recently, single-cell RNA sequencing in the myometrium and MED12 mutation carrying fibroids has identified further heterogeneity in smooth muscle cells and fibroblast cells, identifying 3 different smooth muscle cell populations and fibroblast cell populations. Although both myometrium and fibroids have similar cellular composition, these cells differs in their transcriptomic profile and have specialized roles, underscoring the complex cellular landscape contributing to fibroid pathogenesis. Furthermore, not all smooth muscle cells in MED12-mutant fibroid carry the MED12 mutation, suggesting that MED12-mutant fibroids might not be monoclonal in nature. This review describes the intricacies of fibroid biology revealed by single-cell RNA sequencing. These advances have identified new cellular targets for potential therapies, provided insights into treatment resistance, and laid the groundwork for more personalized approaches to fibroid management. As we continue to unravel the cellular and molecular complexity of fibroids, we anticipate that this knowledge will translate into more effective and less invasive treatments, ultimately improving outcomes for the millions of women affected by this condition.

Chromosomal instability (CIN) is pervasive in human tumours and often leads to structural or numerical chromosomal aberrations. Somatic structural variants (SVs) are intimately related to copy number alterations but the two types of variant are often studied independently. Additionally, despite numerous studies on detecting various SV patterns, there are still no general quantitative models of SV generation. To address this issue, we develop a computational cell-cycle model for the generation of SVs from end-joining repair and replication after double-strand break formation. Our model provides quantitative information on the relationship between breakage fusion bridge cycle, chromothripsis, seismic amplification, and extra-chromosomal circular DNA. Given whole-genome sequencing data, the model also allows us to infer important parameters in SV generation with Bayesian inference. Our quantitative framework unifies disparate genomic patterns resulted from CIN, provides a null mutational model for SV, and reveals deeper insights into the impact of genome rearrangement on tumour evolution.

Sarcomas are rare malignant tumors of mesenchymal origin with a high misdiagnosis rate due to their heterogeneity and low incidence. Conventional diagnostic techniques, such as Fluorescence In Situ Hybridization (FISH) and Next-Generation Sequencing (NGS), have limitations in detecting structural variations (SVs), copy number variations (CNVs), and predicting clinical behavior. Optical genome mapping (OGM) provides high-resolution genome-wide analysis, improving sarcoma diagnosis and prognosis assessment. This study analyzed 53 sarcoma samples using OGM. Ultra-high molecular weight (UHMW) DNA was extracted from core and resection biopsies, and data acquisition was performed with the Bionano Saphyr platform. Bioinformatic pipelines identified structural variations, comparing them with known alterations for each sarcoma subtype. OGM successfully analyzed 62.3% of samples. Diagnostic-defining alterations were found in 95.2% of cases, refining diagnoses and revealing novel oncogenic and tumor suppressor gene alterations. The challenges included DNA extraction and quality issues from some tissue samples. Despite these limitations, OGM proved to be a powerful diagnostic and predictive tool for bone and soft tissue sarcomas, surpassing conventional methods in resolution and scope, enhancing the understanding of sarcoma genetics, and enabling better patient stratification and personalized therapies.

Therapy resistance in breast cancer is increasingly attributed to polyploid giant cancer cells (PGCCs), which arise through whole genome doubling and exhibit heightened resilience to standard treatments. Characterized by enlarged nuclei and increased DNA content, these cells tend to be dormant under therapeutic stress, driving disease relapse. Despite their critical role in resistance, strategies to effectively target PGCCs are limited, largely due to the lack of high-throughput methods for assessing their viability. Traditional assays lack the sensitivity needed to detect PGCC-specific elimination, prompting the development of novel approaches. To address this challenge, we developed a high-throughput single-cell morphological analysis workflow designed to differentiate compounds that selectively inhibit non-PGCCs, PGCCs, or both. Using this method, we screened a library of 2726 FDA Phase 1-approved drugs, identifying promising anti-PGCC candidates, including proteasome inhibitors, FOXM1, CHK, and macrocyclic lactones. Notably, RNA-Seq analysis of cells treated with the macrocyclic lactone Pyronaridine revealed AXL inhibition as a potential strategy for targeting PGCCs. Although our single-cell morphological analysis pipeline is powerful, empirical testing of all existing compounds is impractical and inefficient. To overcome this limitation, we trained a machine learning model to predict anti-PGCC efficacy in silico, integrating chemical fingerprints and compound descriptions from prior publications and databases. The model demonstrated a high correlation with experimental outcomes and predicted efficacious compounds in an expanded library of over 6,000 drugs. Among the top-ranked predictions, we experimentally validated five compounds as potent PGCC inhibitors using cell lines and patient-derived models. These findings underscore the synergistic potential of integrating high-throughput empirical screening with machine learning-based virtual screening to accelerate the discovery of novel therapies, particularly for targeting therapy-resistant PGCCs in breast cancer.

Metastatic cancers arise from a decades-long succession of increasingly virulent precursor lesions, each of which represents prospective targets for therapeutic intervention. This evolutionary process has been particularly vivid in esophageal adenocarcinoma (EAC), as this cancer and associated precursor lesions, including Barrett's esophagus (BE), low-grade dysplasia (LGD), and high-grade dysplasia (HGD), coexist in an accessible, 2-dimensional pattern in esophageal mucosa. Given the durability of these precursor lesions, it is likely that they, like EAC, rely on stem cells for their regenerative growth. To assess the role of stem cells in the evolution of EAC, we apply technology that selectively clones stem cells from the gastrointestinal tract to patient-matched endoscopic biopsies from each of the precursor lesions implicated in EAC.

Histologically validated, endoscopic biopsy series including EAC, HGD, LGD, BE, and normal esophageal mucosa were obtained from patients presenting with EAC. Rare (1:1000) cells from each of these lesions proved clonogenic and were assessed by in vitro differentiation, tumorigenicity in mice, and by molecular genetics.

Each of the lesions in the evolution of EAC possesses a discrete set of clonogenic cells marked by immaturity, enormous proliferative potential, and lesion-specific differentiation fate. DNA sequencing of these clones reveals intralesional heterogeneity and clonal resolution of the mutation progression within a given patient from BE, LGD, HGD, and EAC. High-throughput chemical screens against BE stem cells reveal drug combinations that are similarly effective against stem cells of LGD, HGD, and EAC.

All lesions in the evolution of EAC possess discrete populations of stem cells that are potential therapeutic targets.

Osteosarcoma is the most prevalent cancer-related bone disease diagnosed in the pediatric age group. The rapid development of metastatic lesions and resistance to chemotherapy remain major mechanisms responsible for the failure of treatments and poor outcome. We established that the expression level of Cysteine-rich protein 61 (CYR61/CCN1) correlates to tumor neo-vascularization and dissemination in preclinical and clinical osteosarcoma samples. The aim of this study was to investigate the CYR61-related mechanisms leading to the acquisition of metastatic capacity by osteosarcoma cells.

Transcriptomic data issued from RNA-seq were subjected to pathways and gene set enrichment analyses. Murine and human cell lines with overexpressed or downregulated C-terminal Binding protein 2 (CtBP2) were established by lentiviral transduction. Cell metabolic activity was assessed by Seahorse XF Analyzer; cell replication rate by BrdU incorporation assay; stemness by clonogenicity assay and RT-qPCR detection of markers; cell migration by wound healing assay and Boyden chambers system; cell invasion using Matrigel coated Boyden chambers or fluorescence microscopy of Matrigel embedded 3D spheroids. FFPE samples derived from syngeneic tumor cells grafts into BALB/c mice were analyzed by IHC. The protein interactome was predicted in silico using the STRING database.

GSEA revealed that CYR61 modulate the transcription process. The in vitro expression level of CtBP2 and Cyr61 correlated positively in a panel of osteosarcoma cell lines. In silico analysis of protein-protein interaction network revealed a link with stemness markers. Variations in CtBP2 expression levels influenced stemness markers expression levels, cell clonogenicity, cell migration, Matrix Metalloproteinase activity and cell invasion. Surprisingly, while induction of CtBP2 expression under CYR61 correlated with the metastatic dissemination process in vivo, it occurred only at the invasive front of tumors. Hypoxic conditions in central tumor region interfered with CtBP2 induction of expression.

Our findings identify for the first time that CtBP2 acts as a required critical inducing factor in the CYR61-related metastatic progression of osteosarcoma, by favoring cell migration and invasiveness. Moreover, we demonstrate that while CtBP2 is a downstream transcriptional target of CYR61 signaling cascade, it occurs only under non-hypoxic conditions. The present study suggests that CtBP2 may represent a potential pivotal target for therapeutic management of metastases spreading in osteosarcoma.

Originating from, but independent of, linear chromosomes, extrachromosomal DNA (ecDNA) exists in a more active state of transcription and autonomous replication. It plays a crucial role in the development of malignancies and therapy resistance. Since its discovery in eukaryotic cells more than half a century ago, the biological characteristics and functions of ecDNA have remained unclear due to limitations in detection methods. However, recent advancements in research tools have transformed ecDNA research. It is believed that ecDNA exhibits greater activity in the abnormal amplification of oncogenes, thereby driving cancer progression through their overexpression. Notably, compared to linear DNA, ecDNA can also function as a genomic element with regulatory roles, including both trans- and cis-acting functions. Its critical roles in tumorigenesis, evolution, progression, and drug resistance in malignant tumors are increasingly recognized. This review provides a comprehensive summary of the evolutionary context of ecDNA and highlights significant progress in understanding its biological functions and potential applications as a therapeutic target in malignant tumors.

Chromoanagenesis (CAG) encompasses a spectrum of catastrophic genomic events, including chromothripsis, chromoanasynthesis, and chromoplexy. We studied CAG in 410 patients with a diagnosis of acute myeloid leukemia (AML), 292 newly diagnosed (ND), and 118 refractory/relapsed, using optical genome mapping. CAG was identified by the presence of clusters (with 10 or more breakpoints) of structural abnormalities and/or segmental copy number alterations within one or more chromosomal regions. CAG was detected in 65 (16%) patients. Compared with patients without CAG, those with CAG showed significantly (p < 0.0001) higher frequencies of highly complex karyotype (92% vs. 11%), monosomal karyotype (88% vs. 12%), extensive clonal heterogeneity (75% vs. 7%), gene amplification (49% vs. 1%), and TP53 deletion/mutation (92% vs. 9%). Overall, CAG was detected in about two-thirds of AML patients who exhibited the abovementioned high-risk cytogenetic abnormalities/karyotype. Among the 42 patients with ND AML and CAG, 36 received treatments and follow-ups, and 28 (78%) had no or only partial response to therapy. Among patients with ND AML, those with CAG had a shorter overall survival than those without CAG (median survival: 5 vs. 14 months, p < 0.0001). However, in multivariate analysis, CAG did not appear to be an independent risk factor for survival. These results indicate that CAG is frequently associated with high-risk chromosomal alterations and genomic instability in AML and may contribute to treatment refractoriness and inferior survival in this subset of AML patients.

The fluorescence in situ hybridization (FISH) is a very important technique, as it can diagnose many genetic disorders and cancers. Molecular cytogenetic analysis (FISH) can diagnose numerical chromosome aberrations, sex chromosomes anomalies, and many genetic disorders.

With the limited number of commercially available probes that do not cover all research needs and the high prices of the commercial probes, our goal is to apply recent technologies to produce FISH probes that can accurately and sensitively diagnose genetic diseases and cancer in Egypt and establishing the inhouse production of different FISH probes. We intend to adhere to the published guidelines and validation procedures to ensure the production of accurate FISH probes for clinical diagnosis.

We used specific DNA segments extracted from BAC clones, and we performed nick translation to label the segment with fluorescence labeled dye. The second method involved the use of specific primers for the centromere of certain chromosomes and using PCR technique for amplification and labeling. The probes were tested on metaphase and interphase cells derived from cultured human peripheral blood samples. We followed standard guidelines to test the adequacy of probe slide hybridization, proper probe localization, probe sensitivity and specificity, probe reproducibility, cut-off values, and overall probe validation.

In this research, we presented the generation of three dual-color probes, each probe has a control locus. We offered three dual-color probes targeted 9p21, Xp21 and 17p13.1 loci. chromosome 9p21probe for diagnosis of structural abnormalities in chromosome 9, the Xp21 to test for structural abnormalities of chromosome X, and the 17p13.1 for TP53 gene to detect the loss of p53. We also produced probes for Down syndrome specific region, Rb gene and centromeres for chromosomes X, 17, and 18.

The produced probes are specific and sensitive and can be produced at the commercial level in the laboratory. The production of FISH probes in Egypt can be used as a powerful diagnostic marker for genetic disorders and cancers and our work can be consider as a base to start national project to produce our needs of FISH probes.

Acetamide is a hepatocarcinogen in rats. We previously revealed that acetamide induces characteristic large micronuclei in rat liver, suggesting the possible involvement of chromosome aberrations in acetamide-induced hepatocarcinogenesis. To elucidate the mechanism of large micronuclei formation, in this study we examined time-dependent changes in rat hepatocytes after administration of acetamide. Male 6-week-old F344 rats were gavaged with a single-dose administration of acetamide. A liver micronucleus test showed large micronuclei formation 48 and 72 h after acetamide administration. Histopathological analysis showed binucleated hepatocytes with a unilateral atrophic nucleus beginning 6 h after acetamide administration, and the number reached a maximum at 24 h. At 48 h, the number of binucleated hepatocytes with an atrophic nucleus decreased, and apoptotic hepatocytes and large micronucleated hepatocytes appeared. The changes in the frequency of these abnormal binucleated hepatocytes demonstrated a transition from atrophic nuclei to large micronuclei. Immunohistopathological examinations of binucleated hepatocytes showed loss of nuclear lamina, accumulation of barrier-to-autointegration factor (BAF) and chromatin condensation with heterochromatinization at the atrophic site of nuclei. Results of a BrdU-labeling assay were negative. The abnormal expression of BAF in morphologically normal nuclei suggested that nuclear envelope aberration in hepatocytes was an initial event of the nuclear atrophy. In addition, lack of involvement of cell division in the nuclear atrophy and large micronucleus formation was also demonstrated by BrdU-labeling assay. Overall, our data suggest that large micronuclei induced by acetamide are formed in binucleated hepatocytes through nuclear atrophy.

Recent research reveals that eukaryotic genomes form circular DNA from all parts of their genome, some large enough to carry whole genes. In organisms like yeast and in human cancers, it is often observed that extrachromosomal circular DNA (eccDNA) benefits the individual cell by providing resources for rapid cellular growth. However, our comprehension of eccDNA remains incomplete, primarily due to their transient nature. Early studies suggest they arise when DNA breaks and is subsequently repaired incorrectly. In this review, we provide an overview of the evidence for molecular mechanisms that lead to eccDNA formation in human cancers and yeast, focusing on nonhomologous end joining, alternative end joining, and homologous recombination repair pathways. Furthermore, we present hypotheses in the form of molecular eccDNA formation models and consider cellular conditions which may affect eccDNA generation. Finally, we discuss the framework for future experimental evidence.

Extrachromosomal DNA (ecDNA) are circular DNA bodies that play critical roles in tumor progression and treatment resistance by amplifying oncogenes across a wide range of cancer types. ecDNA lack centromeres and are thus not constrained by typical Mendelian segregation, enabling their unequal accumulation within daughter cells and associated increases in copy number. Despite intrinsic links to their oncogenic potential, the fidelity and mechanisms of ecDNA inheritance are poorly understood. Here, we show that ecDNA are protected against cytosolic mis-segregation through mitotic clustering and by tethering to the telomeric and subtelomeric regions of mitotic chromosomes. ecDNA-chromosome tethering depends on BRD4 transcriptional co-activation and mitotic transcription of the long non-coding RNA PVT1 , which is co-amplified with MYC in colorectal and prostate cancer cell lines. Disruption of ecDNA-chromosome tethering through BRD4 inhibition, PVT1 depletion, or inhibiting mitotic transcription results in cytosolic mis-segregation, ecDNA reintegration, and the formation of homogeneously staining regions (HSRs). We propose that nuclear inheritance of ecDNA is facilitated by an RNA-mediated physical tether that links ecDNA to mitotic chromosomes and thus protects against cytosolic mis-segregation and chromosomal integration.

Chromothripsis is a mutational phenomenon in which a single catastrophic event generates extensive rearrangements of one or a few chromosomes. This extreme form of genome instability has been detected in 30-50% of cancers. Studies conducted in the past few years have uncovered insights into how chromothripsis arises and deciphered some of the cellular and molecular consequences of chromosome shattering. This Review discusses the defining features of chromothripsis and describes its prevalence across different cancer types as indicated by the manifestations of chromothripsis detected in human cancer samples. The different mechanistic models of chromothripsis, derived from in vitro systems that enable causal inference through experimental manipulation, are discussed in detail. The contribution of chromothripsis to cancer development, the selective advantages that cancer cells might gain from chromothripsis, the evolutionary trajectories of chromothriptic tumours, and the potential vulnerabilities and therapeutic opportunities presented by chromothriptic cells are also highlighted.

Osteosarcoma (OS) is a serious malignancy affecting children and young adults; however, there is limited improvement in the survival of patients with OS over the past four decades. Molecular targeted therapy is a promising treatment strategy for OS. Apurinic/apyrimidinic exonuclease 1 (APEX1)-a key factor for DNA damage repair-is associated with OS proliferation, but the underlying molecular mechanism remains unclear. APEX1 expression in OS tissues and paired paracancerous tissues and in human osteoblast cell line hFOB1.19 and OS cell lines was determined using real-time quantitative PCR (RT-qPCR). APEX1-shRNA and NC-shRNA lentiviral vectors were constructed and transfected into MG-63 cells. The effects of APEX1 knockdown on MG-63 cell proliferation and apoptosis were assessed using MTT, xenograft tumor growth, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Expression changes of apoptosis- and angiogenesis-related genes due to APEX1 knockdown were detected using RT-qPCR and immunohistochemistry. To preliminarily determine the mechanism by which APEX1 affects OS cell proliferation, transcription factors were predicted using three databases, and construction of protein-protein interaction network, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed. APEX1 expression was higher in OS tissues than in paracancerous tissues. APEX1 expression was also higher in OS cell lines than in hFOB1.19 cells, with the highest APEX1 expression observed in MG-63 cells. APEX1 knockdown mediated by APEX1-shRNA lentivirus markedly suppressed MG-63 cell proliferation both in vitro and in vivo and induced their apoptosis. APEX1 knockdown downregulated CD31 expression but had no effect on the expression of P53 and Caspase3. Bioinformatics analyses suggested that USF1 or SP1 regulates APEX1 transcription and its recruitment in DNA damage response pathways, affecting OS cell proliferation. Thus, high APEX1 expression in OS facilitates cell proliferation likely via CD31, and USF1 or SP1 may regulate APEX1 transcription and its recruitment in DNA damage response pathways.

We lack tools to edit DNA sequences at scales necessary to study 99% of the human genome that is noncoding. To address this gap, we applied CRISPR prime editing to insert recombination handles into repetitive sequences, up to 1697 per cell line, which enables generating large-scale deletions, inversions, translocations, and circular DNA. Recombinase induction produced more than 100 stochastic megabase-sized rearrangements in each cell. We tracked these rearrangements over time to measure selection pressures, finding a preference for shorter variants that avoided essential genes. We characterized 29 clones with multiple rearrangements, finding an impact of deletions on expression of genes in the variant but not on nearby genes. This genome-scrambling strategy enables large deletions, sequence relocations, and the insertion of regulatory elements to explore genome dispensability and organization.

Cell-cell fusion is a tightly controlled process in the human body known to be involved in fertilization, placental development, muscle growth, bone remodeling, and viral response. Fusion between cancer cells results first in a whole-genome doubled state, which may be followed by the generation of aneuploidies; these genomic alterations are known drivers of tumor evolution. The role of cell-cell fusion in cancer progression and treatment response has been understudied due to limited experimental systems for tracking and analyzing individual fusion events. To meet this need, we developed a molecular toolkit to map the origins and outcomes of individual cell fusion events within a tumor cell population. This platform, ClonMapper Duo ('CMDuo'), identifies cells that have undergone cell-cell fusion through a combination of reporter expression and engineered fluorescence-associated index sequences paired to randomly generated nucleotide barcodes. scRNA-seq of the indexed barcodes enables the mapping of each set of parental cells and fusion progeny throughout the cell population. In triple-negative breast cancer cells CMDuo uncovered subclonal transcriptomic hybridization and unveiled distinct cell-states which arise in direct consequence of homotypic cell-cell fusion. CMDuo is a platform that enables mapping of cell-cell fusion events in high-throughput single cell data and enables the study of cell fusion in disease progression and therapeutic response.

Osteosarcoma is the most common primary cancer of the bone, with a peak incidence in children and young adults. Using multi-region whole-genome sequencing, we find that chromothripsis is an ongoing mutational process, occurring subclonally in 74% of osteosarcomas. Chromothripsis generates highly unstable derivative chromosomes, the ongoing evolution of which drives the acquisition of oncogenic mutations, clonal diversification, and intra-tumor heterogeneity across diverse sarcomas and carcinomas. In addition, we characterize a new mechanism, termed loss-translocation-amplification (LTA) chromothripsis, which mediates punctuated evolution in about half of pediatric and adult high-grade osteosarcomas. LTA chromothripsis occurs when a single double-strand break triggers concomitant TP53 inactivation and oncogene amplification through breakage-fusion-bridge cycles. It is particularly prevalent in osteosarcoma and is not detected in other cancers driven by TP53 mutation. Finally, we identify the level of genome-wide loss of heterozygosity as a strong prognostic indicator for high-grade osteosarcoma.

The heterogeneity and evolution of tumors remain significant obstacles in cancer treatment, contributing to both therapy resistance and relapse. Mesenchymal stem/stromal cells (MSCs) are multipotent stromal cells within the tumor microenvironment that interact with tumor cells through various mechanisms, including cell fusion. While previous research has largely focused on the effects of MSC-tumor cell fusion on tumor proliferation, migration, and tumorigenicity, emerging evidence indicates that its role in tumor maintenance, evolution, and recurrence, particularly under stress conditions, may be even more pivotal. This review examines the connection between MSC-tumor cell fusion and several critical factors like tumor heterogeneity, cancer stem cells, and therapy resistance, highlighting the crucial role of cell fusion in tumor survival, evolution, and recurrence. Additionally, we explore potential therapeutic strategies aimed at targeting this process.

The biomedical community is increasingly invested in capturing all genetic variants across human genomes, interpreting their functional consequences and translating these findings to the clinic. A crucial component of this endeavour is the discovery and characterization of structural variants (SVs), which are ubiquitous in the human population, heterogeneous in their mutational processes, key substrates for evolution and adaptation, and profound drivers of human disease. The recent emergence of new technologies and the remarkable scale of sequence-based population studies have begun to crystalize our understanding of SVs as a mutational class and their widespread influence across phenotypes. In this Review, we summarize recent discoveries and new insights into SVs in the human genome in terms of their mutational patterns, population genetics, functional consequences, and impact on human traits and disease. We conclude by outlining three frontiers to be explored by the field over the next decade.

Chromothripsis, a hallmark of cancer, is characterized by extensive and localized DNA rearrangements involving one or a few chromosomes. However, its genome-wide frequency and characteristics in urothelial carcinoma (UC) remain largely unknown. Here, by analyzing single-regional and multi-regional whole-genome sequencing (WGS), we present the chromothripsis blueprint in 488 UC patients. Chromothripsis events exhibit significant intertumoral heterogeneity, being detected in 41% of UC patients, with an increase from 30% in non-muscle-invasive disease (Ta/1) to 53% in muscle-invasive disease (T2-4). The presence of chromothripsis correlates with an unstable cancer genome and poor clinical outcomes. Analysis of multi-regional WGS data from 52 patients revealed pronounced intratumoral heterogeneity with chromothripsis events detectable only in specific tumor regions rather than uniformly across all areas. Chromothripsis events evolve under positive selection and contribute to tumor dissemination. This study presents a comprehensive genome-wide chromothripsis landscape in UC, highlighting the significance of chromothripsis in UC development.