Background Upper tract urothelial carcinoma (UTUC) is an uncommon but aggressive malignancy. Muscle invasion is strongly associated with poor prognosis and limited treatment options. Revealing the molecular basis of muscle invasiveness in UTUC is crucial. Methods We characterised somatic structural variants (SSVs) from 162 patients with UTUC and integrated these data with bulk RNA-seq, single cell RNA-seq and spatial transcriptomics, with selected SSVs validated by single-molecule sequencing. Furthermore, functional validation experiments, including dual-luciferase reporter assays, wound healing, and invasion/migration assays, were conducted on both the SSV region upstream of TPX2 and the TPX2 gene itself in the 5637 cell line. Findings We found that SSVs were enriched in muscle-invasive (MI) -UTUC compared with non-muscle-invasive (NMI)-UTUC, and were associated with gene expression changes independent of copy-number variation. TPX2 emerged as a representative target, with TPX2-associated SSVs linked to TPX2 over expression and patient poorer progression-free and overall survival. Functional study indicated that a TPX2-upstream SSV region enhances TPX2 expression through promoter regulation, leading to increased invasiveness of 5637 cells. Single cell analyses revealed that TPX2-positive cycling epithelial cells exhibited heightened proliferative signalling and distinct ligand-receptor interactions, particularly involving EGFR- and EPHA2-related pathways. Spatial transcriptomics further localised TPX2-positive spots to epithelial mesenchymal transition (EMT) -enriched neighbourhoods with elevated EGFR/EPHA2 associated ligands, and showed markedly higher abundance in MI-UTUC. Interpretation SSVs drive transcriptional reprogramming, disease aggressiveness and microenvironmental remodelling in UTUC. The signatures of SSVs could classify UTUC into classes with different prognosis. Furthermore, SSVs of TPX2 might be a potential biomarker and candidate therapeutic vulnerability. Funding Stated in acknowledgements section of manuscript.

Intratumoral microbiota play key roles in cancer but are challenging to profile due to low biomass. We developed CIRCMIP, an enzymatic pipeline that eliminates linear DNA to enrich bacterial genomes, achieving 100-fold higher sensitivity than standard metagenomics. Applied to 312 pan-cancer specimens, CIRCMIP identified Staphylococcus lugdunensis as a signature bacterium enriched in early-stage bladder cancer (BLCA), where its presence predicts poor survival. Integrated modeling and lipidomics revealed that S. lugdunensis colonization drives aberrant lipid metabolism with secretion of LPC14:0. Both S. lugdunensis and LPC14:0 drive BLCA progression by promoting fatty acid uptake and β-oxidation. Mechanistically, chemical proteomics revealed LPC14:0 as a direct PPARδ ligand, binding via hydrogen bonds with Thr292/Thr289. This activation upregulates fatty acid transporters (CD36, FABP4) and metabolic enzymes (ACOX2), fueling malignant proliferation. Furthermore, CIRCMIP-derived biomarkers show robust diagnostic accuracy, establishing a new research paradigm and revealing the S. lugdunensis–LPC14:0–PPARδ axis as a therapeutic target in bladder cancer.

Despite representing approximately 30

Extrachromosomal DNA (ecDNA) has emerged as a major driver of genomic instability and rapid tumour evolution in urothelial carcinoma. In urothelial carcinoma, ecDNA amplifies oncogenes, reshapes 3D chromatin interactions, reprogrammes transcription and modulates the tumour-immune interface. Together, these features fuel intratumour heterogeneity, accelerate APOBEC3-associated mutational evolution and contribute to aggressive disease. Advances in sequencing and imaging technologies have greatly expanded our understanding of ecDNA biology. Importantly, ecDNA can be detected through non-invasive liquid biopsies, including urine and plasma, and inferred from standard histopathology slides via digital pathology. These observations suggest that ecDNA could be a valuable adjunct biomarker, enhancing current strategies for early detection, patient stratification and dynamic monitoring of treatment response.

Ureteral carcinoma, a rare malignancy typically presenting as a solitary ureteral lesion. Early diagnosis is challenging due to nonspecific clinical symptoms and subtle radiographic findings. We describe a 41-year-old male with an unusually extensive ureteral tumor that nearly occupied the full length of the right ureter. Multi-regional genomic sequencing demonstrated a branched evolutionary pattern and a complex mutational landscape.

The rapid advancement of DNA foundation language models has brought about a transformative shift in genomics, allowing for the deciphering of intricate patterns and regulatory mechanisms embedded within DNA sequences. The genomic foundation model Evo2 demonstrates remarkable capabilities in decoding DNA functional patterns through cross-species pretraining. However, despite the great potential of Evo2 in basic genomics research, there is currently no clear and systematic guidance on its specific application scenarios, performance, and optimization directions in the field of tumor genomics, and its performance dependency on specialized hardware (such as FP8 precision on H800 GPUs) has not been empirically benchmarked. Here, we present a focused validation of Evo2 using two independent cancer genomic datasets (Bladder Urothelial Carcinoma and Ovarian Cancer), we tested the downstream tasks of Evo2, including the prediction of tumor pathogenic variants and the prediction of mutational effects, and compared its performance on A100 and H800 GPUs. The results show that critical importance of FP8 precision, enabling the H800 to achieve a 4× faster inference speed than the A100 with stable accuracy (AUC 0.88-0.95). The 7B-parameter model emerged as the top performer, whereas the 40B model experienced a severe performance drop (AUC to 0.48) on non-FP8 hardware like the A100. These findings empirically validated Evo2’s hardware specifications and provided practical insights for researchers implementing the model with similar computational resources. Futhermore, our findings provide a framework for the application and optimization of downstream tasks of the DNA language model Evo2 in cancer, and can guide researchers in effectively applying it in genomic studies. Key Points ### Competing Interest Statement The authors have declared no competing interest.

Megabase-sized extrachromosomal circular DNA with intact oncogenes (ecDNA) plays crucial roles in cancer. However, the impact of smaller (100 bp-1 Mb), more ubiquitous extrachromosomal circular DNA (eccDNA) on tumor pathology is unclear. We analyze eccDNA from 122 renal tumors and adjacent tissues, finding increased eccDNA in late-stage cancers, correlating with heightened patient mortality and copy-number amplification. Large eccDNAs are rare, and smaller eccDNAs predominate. The microRNA (miRNA) genes MIR107, MIR196a, MIR495, and MIR519 are recurrently found on eccDNA, almost exclusively in tumors, and patients with any one of these exhibited shorter progression-free survival. Synthetic eccDNAs with these MIR genes increase cancer cell proliferation in 786-O cells, suggesting oncogenic potential. Additionally, we found eccDNA carrying full protein-coding genes that are overexpressed in transcriptional analyses. Our findings suggest that small eccDNAs with miRNA genes are functional and contribute to the tumorigenesis of renal cell carcinoma.

Although assays for microRNA (miRNA) detection have been largely developed and applied, the identification of single-nucleotide polymorphisms in miRNAs (miR-SNPs), which are emerging cancer biomarkers, remains a significant challenge. The rs11614913 SNP in miR-196a2, resulting in the mutant miR-196a2T variant with a single nucleotide change from the wildtype miR-196a2C, has been linked to cancer diagnosis. In this work, we present the split activator ligation assay (SALA) for miR-SNP genotyping, marking the first use of CRISPR/Cas12a in this context. In SALA, miR-196a2T facilitates the ligation of split Cas12a activators via SplintR ligase, leading to the full activation of Cas12a and subsequent fluorescence generation. SALA detects miR-SNPs at femtomolar concentrations, allowing for the precise detection of SNPs when the wildtype allele is in over 2000-fold excess. Analyses of clinical samples demonstrate the robustness of SALA in distinguishing cancerous from non-cancerous samples, underscoring its potential utility in cancer diagnostics.

Background:Extrachromosomal circular DNA (eccDNA) is emerging as a novel biomarker for multiple diseases, but its role in reproductive diseases remains underexplored. Preeclampsia (PE) is associated with increased maternal and perinatal morbidity and mortality. Identifying new screening biomarkers may improve PE-affected pregnancies' outcomes. Methods:To investigate eccDNAs in PE, we collected plasma biopsies from 24 PE-affected and 23 normotensive (Norm) pregnancies, including 14 paired samples collected before and after diagnosis. eccDNA was profiled through linear DNA removal, circular DNA enrichment, and sequencing. The reliability of eccDNA detection was validated using Integrative Genomics Viewer plots and Sanger sequencing. Results:Our analysis identified significantly more eccDNAs in the plasma of PE-affected pregnancies than that in Norm pregnancies (p = 1.67 × 10-6). Notably, eccDNAs in PE were significantly enriched for genes previously found to be associated with PE, including COMT, RORC, CAPZA1, PPP1R12C, AIRE, CYP11A1, CYP11B1, and HSD11B2. Additionally, eccDNA carries four of these genes (AIRE, CYP11A1, CYP11B1, and HSD11B2) are associated with decreased circulating aldosterone levels, a known endocrine feature of PE. Statistically, COMT-containing eccDNA was the most abundant and recurrent in PE. Among the 14 paired plasma samples from pre-diagnosis and diagnosed PE cases, we observed a significant increase in eccDNA abundance in PE (p = 4.14 × 10-10), with 7 of the 9 gene-carrying eccDNAs uniquely identified in PE plasma. Conclusions:This study presents the first eccDNA landscape in the plasma of PE and indicates that plasma eccDNA may serve as a distinguishing hallmark between PE-affected and Norm pregnancies.

Adenosine-to-inosine (A-I) RNA editing is one of the most abundant post-transcriptional RNA modification processes. However, the roles of A-I RNA editing in the evolution and functions of primate brains are underexplored. Here, we perform whole-genome and whole-transcriptome sequencing of 39 anatomically defined brain regions of adult Macaca fascicularis and identify 2 782 079 A-I editing sites, including 2009 recoding sites enriched in genes related to neurotransmission functions. Most of macaque brain A-I editing sites are detected in the cerebral cortex, cerebellum, and amygdala. The brain A-I editing activity is associated with the estimated proportion of neurons to some extent. Comparison of A-I editing in mouse, pig, macaque, and human brains reveals that primates exhibit higher editing levels, specifically enriched in genes encoding neurotransmitter receptors. We identify 478 598 conserved brain editing sites between human and macaque brains, mostly found in the cerebral cortex and enriched in genes related to the cytoskeletal system and ubiquitin-dependent protein degradation pathway. Our study sheds light on the importance of post-transcriptional A-I RNA editing in the evolution and function of nervous systems.

Extrachromosomal DNA (ecDNA) presents a promising target for cancer therapy; however, its spatial-temporal diversity and influence on tumor evolution and the immune microenvironment remain largely unclear. We apply computational methods to analyze ecDNA from whole-genome sequencing data of 595 urothelial carcinoma (UC) patients. We demonstrate that ecDNA drives clonal evolution through structural rearrangements during malignant transformation and recurrence of UC. This supports a model wherein tumors evolve via the selective expansion of ecDNA-bearing cells. Through multi-regional sampling of tumors, we demonstrate that ecDNA contributes to the evolution of multifocality and increased intratumoral heterogeneity. EcDNA is present in 36% of UC tumors and correlates with an immunosuppressive phenotype and poor prognosis. Single-cell RNA sequencing analyses reveal that ecDNA+ malignant cells exhibit diminished expression of major histocompatibility complex class I molecules, enabling them to evade T-cell immunity. Finally, we show that sequencing of urinary sediment-derived DNA has excellent specificity in detecting ecDNA.

Dear Editor, The accurate diagnosis of cancerous nodules in nodular thyroid disease remains a significant challenge.1, 2 Extrachromosomal circular DNA (eccDNA), generated during apoptosis, shows tissue/plasma-specific patterns, and varying spectra in different diseases. However, its role in distinguishing benign and malignant thyroid nodules is unexplored.3-9 This study leverages Circle-seq technology and machine learning to investigate the potential of eccDNA as a non-invasive biomarker for diagnosing thyroid cancer. To understand the distribution of eccDNA in plasma amongst healthy controls (NOR, N = 13), nodular thyroid goitre patients (NOD, N = 25) and papillary thyroid carcinoma (PTC, N = 47) individuals, we applied an optimised Circle-seq strategy for enriching circular DNA (Figure 1A), with the clinical characteristics of the subjects detailed in Table S1. The majority of the Circle-seq reads were aligned to the human reference genome (Figure S1). We examined the eccDNA profiles in healthy controls and NOD/PTC patients, noting significant variability in eccDNA counts amongst groups. On average, NOR, NOD and PTC groups harboured 27 019 (range: 11 822–45 622), 16 850 (range: 7632–44 537) and 27 352 (range: 4015–83 301) unique eccDNAs, respectively. After normalising eccDNA counts to eccDNA counts per million mapped reads (EPM) to adjust for sequencing depth variations, the PTC group showed significantly elevated EPM values compared to both NOD and healthy controls (Figure 1B). Consistent with increased EPM values in PTC, this group also exhibited a higher number and proportion of eccDNA mapping to protein-coding regions, while repeat element proportions were lower (Figure 1C–E). EccDNA load variations prompted us to explore its relationship with cancer characteristics, revealing that loss of eccDNA from coding regions correlates positively with tumour size, suggesting the specific roles of eccDNA in oncogenesis (Figure S2). The plasma eccDNA analysis in the PTC group showed longer fragments and higher Guanine-Cytosine (GC) content, with most eccDNA populations under 1000 bases, peaking at around 202 and 338 bases, a preference for high GC content areas in eccDNA formation (Figure S3A–C). Genomic annotation highlighted eccDNA's enrichment in untranslated and exonic regions, aligning with known profiles in health and disease (Figure 1F). To understand the distribution of eccDNA genomic locations in PTC patients, we aligned eccDNA sequences with the human genome and identified 450 gene-containing eccDNA (eccGenes) that were significantly prevalent in the PTC group (Figure S4A). Gene ontology (GO) and gene set enrichment analysis (GSEA) indicated that these eccGenes frequently involve exons related to tissue growth, and the Wnt and GPCR signalling pathways (Figure S4B,C). Principal component analysis (PCA) highlighted distinct eccDNA gene region diversity associated with PTC (Figure 1G). Remarkably, an increased presence of miR-1203-related eccDNA circles was detected in the PTC group (Figure 2A,B). Transfection of synthesised miR-1203 eccDNA into thyroid cell lines (TPC-1, BHP10-3 and K1) led to significant transcriptional changes: 572 genes were upregulated and 1035 downregulated (Figure 2C). This significant shift in gene expression, involving numerous cancer-associated genes, underscores eccDNA's influence in oncogenesis (Figure 2D,E). To investigate if PTC tumour cells emit eccDNA into the bloodstream, we crafted mouse xenograft models with human PTC cell lines TPC-1, BHP10-3 and K1. We isolated eccDNA from plasma, confirming human-origin eccDNA in mice (Figure 3A). Sequencing revealed genomic features consistent with tumour-derived eccDNA, including GC content, motif patterns and chromosomal distribution, highlighting its potential as a cancer detection marker (Figure 3B–D). Exploring eccDNA's diagnostic value in PTC, we analysed 308 837 genomic locations. Utilising an E-net logistic regression model and a two-nested leave-one-out strategy,10 we assessed the link between these locations and disease status. We identified 71 critical eccDNA locations capable of differentiating PTC from NOD patients, forming a potential classification model. By comparing 71 eccDNA locations identified in this study with the gene annotations of the human genome GRCh38.p13 in Ensembl (version 108), we found 18 locations showed no overlap with any known genes, while the remaining 53 locations overlapped with 71 known genes (Figure 4A and Table S2). After completing the two-nested leave-one-out cross-validation (LOOCV) process, we generated 72 predictions for the 72 subjects. Receiver operating characteristic curve (ROC) analysis on these predictions and their true labels produced an area under the curve (AUC) of .754 (Figure 4B). To further evaluate the diagnostic efficacy of our model, we have supplemented it with more detailed performance metrics, including an accuracy of 68.1%, sensitivity of 55.3%, specificity of 92.0%, positive predictive value of 92.9% and negative predictive value of 52.3%. We also selected the optimal cut-off point using a two-nested LOOCV approach. To address possible bias from random training and validation set splits, we conducted 2000 rounds of five-fold cross validation, averaging an AUC of .789 with a standard deviation of .135. Additionally, a T-test comparing predictions across the two groups indicated a statistically significant difference (T = −3.9012, p = .00029; Figure 4C), suggesting the 71 identified eccDNA locations could serve as a diagnostic biomarker for distinguishing between PTC and NOD patients. This study explores the potential of plasma eccDNA as a non-invasive biomarker to distinguish between benign and malignant thyroid nodules, potentially improving the diagnosis of PTC. The elevated eccDNA levels found in PTC patients and mouse PTC xenograft models indicate its tumour-originated release into circulation, supporting its diagnostic value. In this study, the sample size is an important limitation, which restricts the exploration of research conclusions and the performance of the model to a certain extent. A small sample size may lead to overfitting and reduce the credibility of research conclusions. To address this, we adopted a two-nested LOOCV loops to ensure that the testing set in the outer loop does not involve repeated training. The results from the outer loop were used as evaluation criteria, maximising data utilisation and preventing overfitting. After our posterior sample size estimation, our sample size meets the minimum sample size requirement, indicating that the research conclusions are credible. In the future, we will expand the sample size to improve our research model and conclusions. Despite limited samples, we identified the nucleosomal origin and genetic predisposition for eccDNA formation in PTC, establishing eccDNA as a promising biomarker for cancer detection and enhancing thyroid cancer diagnostics precision. Jiajun Zhao, Yonglun Luo, Ling Gao and Changbin Yu conceived the idea. Meng Zhou, Zhenyu Yao and Yunyun Xu collected the clinical samples and constructed the mouse xenograft models. Wei Lv and Peng Han performed the Circle-seq experiments. Yonglun Luo, Wei Lv, Meng Zhou, Peng Han, Kai Sun and Ziqian Hao drafted most of the manuscript. Wei Lv, Peng Han and Zhe Xu analysed the eccDNA data. Kai Sun, Ziqian Hao and Meng Zhou encoded the eccDNA sequences and performed machine learning and statistical analysis. Shanshan Shao, Shizhan Ma, Qingling Guo, Haiqing Zhang, Ke Liu, Fan Yang, Zhongshang Yuan, Jiajun Zhao and Guojun Wu have contributed to the execution of the experiments and studies. Meng Zhou, Wei Lv, Peng Han, Kai Sun, Ziqian Hao, Guojun Wu, Changbin Yu, Zhenyu Yao, Ling Gao and Jiajun Zhao discussed the results and contributed to the final manuscript. This work was supported by the National Key Research and Development Program of China (2021YFA0805100), Shandong Provincial Project (ZFJH202306) and the National Natural Science Foundation of China (81430020, 81900717, 82070818 and 82200879). The authors express their gratitude to Prof. Christopher G. Proud and Prof. Xuemin Wang for their valuable comments and suggestions during the writing process. We extend our thanks to Lifang Zhao, Jin Xie, Bo Xiang, Ning Wang, Xiaoqing Sun and Wenbo Bian for their technical assistance. We are also appreciative of Xiuyun Li and Jindi Zhang for the clinical samples collection. Our gratitude goes to Figdraw2.0 (www.figdraw.com) for figures generation. The authors declare no conflicts of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract Bladder carcinoma (BC) accounts for > 90% of all urothelial cancers. Pathological diagnosis through cytoscopic biopsy is the gold standard, whereas non-invasive diagnostic tools remain lacking. The “Atyp.C” parameter of the Sysmex UF-5000 urine particle analyzer represents the ratio of nucleus to cytoplasm and can be employed to detect urinary atypical cells. The present study examined the association between urinary Atyp.C values and BC risk. This two-center, retrospective case–control study identified clinical primary or newly recurrent BC (study period, 2022–2023; n = 473) cases together with controls with urinary tract infection randomly matched by age and sex (1:1). Urinary sediment differences were compared using non-parametric tests. The correlations between urinary Atyp.C levels and BC grade or infiltration were analyzed using Spearman’s rank correlation. The BC risk factor odds ratio of Atyp.C was calculated using conditional logistic regression, and potential confounder effects were adjusted using stepwise logistic regression (LR). Primary risk factors were identified by stratified analysis according to pathological histological diagnosis. The mean value of urinary Atyp.C in BC cases (1.30 ± 3.12) was 8.7 times higher than that in the controls (0.15 ± 0.68; P < 0.001). Urinary Atyp.C values were positively correlated with BC pathological grade and invasion (r = 0.360, P < 0.001; r = 0.367, P < 0.001). Urinary Atyp.C was an independent risk factor for BC and closely related with BC pathological grade and invasion. Elevated urinary Atyp.C values was an independent risk factor for BC. Our findings support the use of Atyp.C as a marker that will potentially aid in the early diagnosis and long-term surveillance of new and recurrent BC cases.

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.

Ribosomal DNA (rDNA) encodes the ribosomal RNA genes and represents an intrinsically unstable genomic region. However, the underlying mechanisms and implications for genome integrity remain elusive. Here, we use Bloom syndrome (BS), a rare genetic disease characterized by DNA repair defects and hyper-unstable rDNA, as a model to investigate the mechanisms leading to rDNA instability. We find that in Bloom helicase (BLM) proficient cells, the homologous recombination (HR) pathway in rDNA resembles that in nuclear chromatin; it is initiated by resection, replication protein A (RPA) loading and BRCA2-dependent RAD51 filament formation. However, BLM deficiency compromises RPA-loading and BRCA1/2 recruitment to rDNA, but not RAD51 accumulation. RAD51 accumulates at rDNA despite depletion of long-range resection nucleases and rDNA damage results in micronuclei when BLM is absent. In summary, our findings indicate that rDNA is permissive to RAD51 accumulation in the absence of BLM, leading to micronucleation and potentially global genomic instability. Ribosomal DNA is an unstable genomic region with the underlying mechanisms remaining elusive. Here the authors find that rDNA hyper recombination in Bloom Syndrome cells arises from RAD51 accumulation in the absence of long-range resection and cause genome instability through micronuclei formation.

IntroductionCARD11 is a lymphoid lineage-specific scaffold protein regulating the NF-κB activation downstream of the antigen receptor signal pathway. Defective CARD11 function results in abnormal development and differentiation of lymphocytes, especially thymic regulatory T cells (Treg).MethodIn this study, we used patients’ samples together with transgenic mouse models carrying pathogenic CARD11 mutations from patients to explore their effects on Treg development. Immunoblotting and a GFP receptor assay were used to evaluate the activation effect of CARD11 mutants on NF-κB signaling. Then the suppressive function of Tregs carrying distinct CARD11 mutations was measured by in vitro suppression assay. Finally, we applied the retroviral transduced bone marrow chimeras to rescue the Treg development in an NF-κB independent manner.Results and discussWe found CARD11 mutations causing hyper-activated NF-κB signals also gave rise to compromised Treg development in the thymus, similar to the phenotype in Card11 deficient mice. This observation challenges the previous view that CARD11 regulates Treg lineage dependent on the NF-kB activation. Mechanistic investigations reveal that the noncanonical function CARD11, which negatively regulates the AKT/ FOXO1 signal pathway, is responsible for regulating Treg generation. Moreover, primary immunodeficiency patients carrying CARD11 mutation, which autonomously activates NF-κB, also represented the reduced Treg population in their peripheral blood. Our results propose a new regulatory function of CARD11 and illuminate an NF-κB independent pathway for thymic Treg lineage commitment.

Rationale: Extrachromosomal circular DNA is a hallmark of cancer, but its role in shaping the genome heterogeneity of urothelial bladder carcinoma (UBC) remains poorly understood. Here, we comprehensively analyzed the features of extrachromosomal circular DNA in 80 UBC patients. Methods: We performed whole-genome/exome sequencing (WGS/WES), Circle-Seq, single-molecule real-time (SMRT) long-read sequencing of circular DNA, and RNA sequencing (RNA-Seq) on 80 pairs of tumor and AT samples. We used our newly developed circular DNA analysis software, Circle-Map++ to detect small extrachromosomal circular DNA from Circle-Seq data. Results: We observed a high load and significant heterogeneity of extrachromosomal circular DNAs in UBC, including numerous single-locus and complex chimeric circular DNAs originating from different chromosomes. This includes highly chimeric circular DNAs carrying seven oncogenes and circles from nine chromosomes. We also found that large tumor-specific extrachromosomal circular DNAs could influence genome-wide gene expression, and are detectable in time-matched urinary sediments. Additionally, we found that the extrachromosomal circular DNA correlates with hypermutation, copy number variation, oncogene amplification, and clinical outcome. Conclusions: Overall, our study provides a comprehensive extrachromosomal circular DNA map of UBC, along with valuable data resources and bioinformatics tools for future cancer and extrachromosomal circular DNA research.

ABSTRACT Extrachromosomal circular DNAs (ecDNA) are focal gene amplifications frequently associated with cancer development and often indicating a poor prognosis. To understand the early dynamics of oncogene-carrying ecDNAs, we previously developed CRISPR-C, a tool for precise ecDNA generation by deleting specific chromosomal regions. Here, we adapted CRISPR-C to recreate tumor ecDNAs. This method also allowed us to enhance ecDNA generation efficiency by directly delivering Cas9 protein and sgRNAs as a ribonucleoprotein complex. By using the modified CRISPR-C, we successfully generated ecDNAs carrying oncogenes ( EGFR, CDK4, MDM2, MYC, MYCN, FGFR2, ABCB1, and DHFR ) in various human cell types. Furthermore, we demonstrated that our method could generate chimeric ecDNAs composed of target sequences from distant intra or inter-chromosomal regions. Using these generative ecDNA cell models, we studied the oncogene ecDNA expression and stability. The MDM2 expression was increased after CRISPR-C, while CDK4 was decreased indicating genomic-context dependent effect. The copy number of CRISPR-C generated CDK4 was ecDNA increased in cells after a long period of treatment with the CDK4 inhibitor palbociclib. Unlike CDK4, the CRISPR-C generated ABCB1 ecDNA was unstable in cells under normal growth conditions, but is stably retained when the cells were treated with colcemid, a recognized substrate for ABCB1. We thus provide valuable tools and an attractive platform for studying ecDNA biogenesisy and in vitro drug screening on ecDNA stability.

Extrachromosomal circular DNA (eccDNA) derived from linear chromosomes, are showed typical nucleosomal ladder pattern in agarose gel which as a known feature of apoptosis and demonstrated to be immunogenicity. In systemic lupus erythematosus (SLE) patients, elevated levels of cell-free DNA (cfDNA) can be found in either linear forms or circular forms, while circular ones are much less common and harder to detect. The molecular characteristics and function of circular forms in plasma SLE patients remains elusive. Herein, we characterized the hallmarks of plasma eccDNA in SLE patients, including the lower normalized number and GC content of eccDNA in SLE plasma than in the healthy, and SLE eccDNA number positively correlated with C3 and negatively with anti-dsDNA antibodies. The differential eccGenes (eccDNAs carrying the protein coding gene sequence) of SLE was significantly enriched in apoptosis-related pathways. The artificially synthesized eccDNA with sequences of the PRF1 exon region could promote transcriptional expression of PRF1, IFNA and IFIT3 and inhibit early-stage apoptosis. Plasma eccDNA can serve as a novel autoantigen in the pathogenesis of SLE.

Ureteral carcinoma, a rare malignancy, is often characterized by the presence of solitary lesions in the ureter. Early diagnosis and effective management are compounded by nonspecific clinical symptoms and radiographic findings. Here, we present a case of a 41-year-old male with a tumor that nearly spanned the entire length of the right ureter. Multi-regional genomic sequencing of the tumor revealed a pattern of branch evolution in this patient, accompanied by a complex genomic mutational landscape.