Extrachromosomal DNA (ecDNA) plays a key role in cancer pathology. EcDNAs mediate high oncogene amplification and expression and worse patient outcomes. Accurately determining the structure of these circular molecules is essential for understanding their function, yet reconstructing ecDNA cycles from sequencing data remains challenging. We introduce Cycle-Extractor (CE) for reconstruction. CE accepts a breakpoint graph derived from either short or long read sequencing data as input and extracts a cycle with the maximum length-weighted-copy-number. CE utilizes a mixed-integer linear program (MILP) and a separate traversal procedure, enabling fast optimization and compatibility with free solvers. We evaluated CE against CoRAL (long-read-based quadratic optimization), Decoil (long-reads), and AmpliconArchitect (AA for short reads) on both simulated data and real cancer cell lines. On simulated ecDNA, CE achieves performance comparable to CoRAL across three accuracy metrics and consistently outperforms AA and Decoil. On cancer cell lines, CE produces longer and heavier cycles than AA, and achieves performance similar to CoRAL. Moreover, CE is, on average, 40× faster than CoRAL. These results demonstrate that CE accurately reconstructs ecDNA from both short- and long-read sequencing data, while long-read inputs allow CE to recover more complete and higher-confidence ecDNA structures. CE improved the prediction of many ecDNA structures. On a PC3 ecDNA containing MYC , CE uses ONT data to reconstruct a substantially larger and higher-copy sequence (4.2 Mbp) compared to the short-read-derived reconstruction (690 Kbp). CRISPR-CATCH experiments confirm the presence of a large ecDNA molecule, validating the long-read-based CE reconstruction.

Abstract Uterine corpus endometrial carcinoma (UCEC) is a common gynecologic malignancy with rising mortality, yet its genome-wide mutational architecture remains incompletely understood. Here we analyze deep whole-genome sequencing data from 440 UCEC tumors from The Cancer Genome Atlas, integrated with transcriptomic and clinical data, to define subtype-specific mutational processes and genomic architectures. We uncover pronounced molecular subtype-specific differences in endogenous mutational mechanisms, retrotransposition activity, structural variation, and chromosomal instability. LINE-1 retrotransposition emerges as a key contributor to genome instability in UCEC, acting as a prominent source of structural variation in copy-number stable tumors and associating with chromothripsis and ecDNA-mediated oncogene amplification in copy-number high tumors. Mutational signature SBS28 contributes significantly to mutations in POLE -deficient tumors and is strongly correlated with SBS10a and SBS10b. Mismatch repair-deficient tumors exhibit a previously unrecognized doublet base substitution signature and a defining imbalance between ID2 and ID1 indel processes, reflecting pervasive DNA template-strand replication slippage and associated with increased tumor immunogenicity. Copy-number low tumors follow a distinct low-mutagenesis evolutionary trajectory characterized by reduced replication stress, low proliferative activity, genomic stability and enrichment of SBS18 associated with oxidative damage. Notably, we identify a consistent inverse association between body mass index and tumor mutational burden in UCEC, suggesting that host metabolic state may influence fundamental cellular processes governing mutation accumulation, promoting tumor development through non-mutagenic mechanisms rather than elevated genomic instability. Together, these findings establish a mutagenesis-centric framework for UCEC that links endogenous mutational mechanisms to tumor architecture and host metabolic context, uncovering previously unrecognized subtype-specific genomic features with important implications for refined risk stratification and therapeutic strategies.

Abstract Gastric adenocarcinoma (GAC) remains a major health challenge with high mortality due to late diagnosis, therapy resistance, and limited targeted options. Extrachromosomal DNA (ecDNA)—circular DNA elements carrying amplified oncogenes such as MYC, ERBB2, and EGFR—enhances transcriptional output, therapy resistance, and genomic instability. However, its role in GAC is poorly understood, and current models fail to recapitulate patient tumor complexity. Patient-derived organoids (PDOs) preserve tumor-specific features, including ecDNA, providing an ideal platform for mechanistic and therapeutic exploration.We hypothesize that ecDNA drives unique oncogenic programs in GAC that create exploitable vulnerabilities. Analysis of 221 TCGA-STAD samples with AmpliconArchitect identified ecDNA in 33.3% of primary tumors, absent in matched normal blood. ecDNA+ tumors showed distinct amplification patterns, with proteomic profiling revealing CHEK1 and TFRC enrichment, suggesting co-targetable vulnerabilities. To model these tumors, we established a PDO biobank comprising 5 primary and 9 ascites-derived PDOs. ecDNA was detected in 5/9 (55.6%) ascites-derived and 1/5 (20%) primary PDOs, consistent with ecDNA enrichment in metastatic settings. Amplified oncogenes included PTP4A3, for which inhibitors are available.RNA-seq comparison of ecDNA+ PDO 385 with ecDNA- PDOs (4666, 4601) revealed distinct transcriptional programs, with enriched expression on chromosomes 8 and 20. Transcription factor analysis identified MYC, also amplified as ecDNA. Immunofluorescence confirmed higher TFRC, CHEK1, MYC, and PRL3 (PTP4A3) expression and increased Ki67 in ecDNA+ PDO 385, supporting oncogene overexpression and aggressive growth. Drug screening predicted compounds reversing the ecDNA+ signature. Functional assays showed the TFRC inhibitor (TfR-1-IN-1) reduced proliferation of ecDNA+ PDO 385, while CHK1 inhibition had modest effects. Combined TFRC and CHK1 inhibition synergistically suppressed ecDNA+ PDO growth, with minimal effects on ecDNA- controls. These results highlight the selective vulnerability of ecDNA-driven tumors to co-targeting strategies. Using a PDO biobank, we aim to uncover and exploit vulnerabilities in ecDNA-positive GAC. Citation Format: Gengyi Zou, Melissa Pizzi, Aryanasingh Bhati, Bansi Vanparia, Wei-Chieh Yu, Ailing Scott, Yibo Fan, Calena Brown-Abel, Liyong Zeng, Johnson Amoah, Tanaya Alexander Washington, Yuan-Hung Lo, Vineet Bafna, Shilpa Dhar, Jaffer Ajani. Combined pan and precision targeting in extrachromosomal DNA-positive gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4858.

Understanding ecological change requires robust, inexpensive methods for monitoring populations. One approach is using low-coverage genome sequencing (genome skimming) to compute genetic diversity via assembly-free, alignment-free, k-mer-based methods to compute genomic distances from shotgun sequences using the intersection of k-mer sets. Skmer extended this approach to genome skims by modeling coverage and error. However, these methods ignore genome repetitiveness, hampering population genetic distance calculation. Here, we mathematically derive the expected intersection size between k-mers sampled from two repetitive genomes, accounting for repeats, coverage, and errors, leading to the method ReSkmer. Our experiments show highly accurate distances despite sampling highly repetitive genomes.

Abstract Introduction: Extrachromosomal DNA (ecDNA) contributes to oncogene amplification in human cancers, yet the mechanisms underlying the formation and function of human-viral hybrid ecDNA (hybrid ecDNA), circular elements harboring both HPV and human sequences, remain unclear. We recently identified hybrid ecDNA in human papillomavirus-associated oropharyngeal cancer (HPVOPC), a rapidly increasing malignancy. Here, we show that de novo enhancer formation at viral-human junctions activates viral oncogenes and promotes hybrid ecDNA self-amplification, revealing a tractable therapeutic vulnerability. Methods: Hybrid ecDNA was identified in HPVOPC cell lines and patient-derived xenografts (PDX) using whole-genome sequencing and AmpliconArchitect. Validation was performed by multicolor FISH. Chromatin accessibility and enhancer activity were profiled by ATAC-seq and H3K27ac ChIP-seq. Hi-C sequencing was used to assess 3D chromatin interactions and potential contacts among distinct ecDNA species. Functional significance was evaluated by CRISPR interference (CRISPRi) targeting ecDNA-derived enhancers. Therapeutic relevance was assessed by BET inhibition in vitro and in vivo. Results: Hybrid ecDNA was detected in both cell lines and PDX tumors and validated by overlapping human and HPV FISH signals. Epigenomic profiling revealed de novo active enhancers flanking HPV sequences exclusively in hybrid ecDNA(+) tumors. Hi-C demonstrated enhancer-promoter loops linking host enhancers to HPV oncogenes. CRISPRi targeting these enhancers significantly inhibited proliferation in hybrid ecDNA(+) models only (P = 0.006). BET inhibition selectively suppressed hybrid ecDNA(+) tumor growth in vivo (P = 2×10-5). Importantly, Hi-C contacts also indicated enriched interaction patterns among distinct ecDNA species, suggesting potential extrachromosomal cooperation that enhances viral oncogene expression. Conclusions: Hybrid ecDNA is a functionally critical structure in HPVOPC by generating de novo enhancers and reorganizing chromatin interactions to activate viral oncogenes, creating a therapeutic vulnerability. Contact enrichment among distinct ecDNA species further suggests participation in a broader extrachromosomal regulatory network in HPV-driven malignancies. Citation Format: Takuya Nakagawa, Jens Luebeck, Kaiyuan Zhu, Sasik Roman, Brin Rosenthal, Kathleen Fisch, Toyoyuki Hanazawa, Atsushi Kaneda, Paul S. Mischel, Vineet Bafna, Joseph A. Califano. De novo enhancer formation at human-viral junctions drives hybrid extrachromosomal DNA amplification in HPV-associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1925.

Abstract Ductal carcinoma in situ is a non-obligate precursor lesion of breast cancer. Often detected by mammography, most cases are managed through surgical and/or radiotherapy approaches. Today, it is not possible to predict which patients will progress to invasive disease. Here, we evaluate high-depth whole-genome sequenced ductal carcinoma in situ, enriched for high-grade clinical lesions, to understand whether deep WGS could reveal biological insights and/or personalized therapeutic vulnerabilities that may be targetable. We find genomic locations that are likely susceptible to producing the initiating lesion for structural variations prone to subsequent evolution, termed SHOREs. We additionally highlight individualized therapeutic potential that would otherwise not be appreciable without whole genome sequencing. We posit that holistic whole genome sequencing profiling could offer a more precise stratification approach, discerning higher-risk cases for prospective clinical studies on personalized therapies, from truly low-risk cases suitable for active monitoring.

Extrachromosomal DNA (ecDNA) amplifications are key drivers of human cancers. Here, we show that ecD-NAs are major platforms for generating and amplifying oncogene fusion transcripts across diverse cancer types. By integrating analysis of whole-genome and transcriptome sequences from tumor samples and cancer cell lines of a wide variety of tissue types, we reveal that ecDNAs have the highest rate of oncogene fusion events of any copy-number alteration. Focusing on the most common ecDNA fusion hotspot, we find that fusion of the 5 ' end of the long noncoding RNA gene, PVT1-with exon 1 joined to diverse 3 ' partners-confers increased RNA stability, potentially via an SRSF1-dependent mechanism, and enhances MYC-dependent transcription and cancer cell survival. These results demonstrate that ecDNA fosters genome instability and frequent oncogene fusion formation in cancer.

Some aggressive cancers exhibit a level of rapid genome change and therapy resistance that is difficult to explain. Research over the past decade has shown that extrachromosomal DNA (ecDNA) can be the cause. When oncogenic genetic elements untether from chromosomes and no longer follow Mendelian inheritance, genomic chaos and accelerated evolution ensue, generating unique ecDNA biology and non-traditional therapeutic vulnerabilities distinct from traditional mutation-targeting approaches. Here, we put forward a holistic view where ecDNA is integrated into the broader Hallmarks of Cancer framework to better understand the problem and chart a path forward.

Abstract Uterine corpus endometrial carcinoma (UCEC) is among the most prevalent gynecologic malignancies worldwide and represents a growing public health burden, particularly in high-income countries. Despite advances in clinical management, UCEC mortality continues to rise, with marked racial and socioeconomic disparities. The Cancer Genome Atlas (TCGA) previously established a molecular framework classifying UCEC into four major subtypes: POLE-ultramutated, microsatellite instability-high (MSI), copy-number low (CN-Low), and copy-number high (CN-High). However, the lack of whole-genome sequencing (WGS) has limited comprehensive characterization of UCEC genomic architecture. Here, we analyzed WGS data from 440 TCGA UCEC tumors, together with matched multi-omics data, to systematically characterize subtype-specific mutational processes, structural variation, and their clinical associations. Among driver genes, POLE-ultramutated tumors exhibited extensive multiple mutations, depleted frameshift indels, and increased subclonal heterogeneity. Across UCEC, LINE-1 retrotransposition events predominantly originated from a germline source at chr22q12.1, with endometrioid-like CN-High tumors displaying the highest level of transposable element activity. CN-High tumors also showed high frequent extrachromosomal DNA (ecDNA) amplification, dominated by co-amplification of the MYC-PVT1 and ERBB2-MIEN1 loci, whereas ecDNA events were rare in other subtypes. Within CN-High tumors, LINE-1 insertion burden was strongly correlated with ecDNA formation, suggesting a mechanistic link between retrotransposition-associated genomic instability and oncogene amplification via ecDNA. In contrast, serous-like CN-Low tumors were characterized by higher patient body mass index (BMI), lower replication stress and increased X-chromosome inactivation. Mutational signature analysis revealed striking subtype specificity. APOBEC signatures were largely restricted to CN-High tumors. SBS39, a previously uncharacterized signature, was highly prevalent in CN-High tumors and significantly associated with BMI. MSI tumors exhibited a novel DBS signature enriched for A/T-containing reversed doublet substitutions (XY→YX), while indel mutagenesis was nearly ubiquitous and restricted to ID1 and ID2 signatures. Notably, the ID2-to-ID1 ratio in MSI tumors was approximately ten-fold higher than in non-MSI tumors, indicating a strong bias toward template-strand replication slippage associated with mismatch repair deficiency. Together, these findings uncover previously unrecognized, subtype-specific genomic features in UCEC and define distinct mutational mechanisms with potential implications for refined risk stratification and therapeutic strategies. Citation Format: Jian Sang, Mengyan Zhang, Thomas Veith, Yewon Kim, Sergio Chavez, Weiyin Zhou, Wen Luo, Adriana Morales Miranda, Jens Luebeck, Vineet Bafna, Stephen J. Chanock, Tongwu Zhang. Whole-genome sequencing reveals distinct mutational mechanisms across endometrial cancer subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB101.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a common driver of tumor progression and is associated with acquired drug resistance and poor patient survival. While whole genome sequencing (WGS) studies have revealed the landscape of genes amplified on ecDNA in tumors, it remains challenging to study the subclonal heterogeneity and functional (e.g., transcriptomic) consequences of ecDNA on tumors. To address this, we introduce scAmp: a probabilistic algorithm for detecting and analyzing ecDNA from single-cell datasets. We demonstrate scAmp's improved accuracy over WGS approaches on well-characterized cell-lines and its applicability to clinical histopathology. We further showcase scAmp by analyzing 73 patient tumors profiled with single-cell ATAC-seq, where we analyze the subclonal evolution of ecDNA+ subclones and identify the effect of ecDNA amplifications on the chromatin accessibility landscape of cancer cells. Together, we anticipate that scAmp will broadly enable further studies - both retrospective and prospective - that dissect critical questions of how ecDNA affect cancer cells and the tumors in which they reside.

Ultraconserved elements (UCEs) provide ideal candidates for targeted sequencing and cost-effective acquisition of genome-wide data. While UCEs have been widely used in phylogenetic studies to reconstruct evolutionary relationships, their use in population-level research has been limited. This limited application stems from uncertainty over whether UCEs can capture the levels of genetic variation needed to answer population genomic questions central to ecology and biodiversity research. The concern is that, by definition, UCEs are highly conserved and may therefore lack sufficient within-species variation. The more variable flanking regions (400-750 bp from the UCE core) contain informative polymorphisms, though diversity decreases near the core. Thus, any naive estimator of genetic diversity that ignores this conservation will have an underestimation bias. In this paper, we introduce SPrUCE: Sigmoid Pi requiring UCEs, a reference-free method that estimates nucleotide diversity π $$ \pi $$ from aligned UCE data. SPrUCE corrects underestimation bias by modelling the change in diversity away from the UCE core using a Gompertz function. The model accounts for the bias introduced by the conserved core and allows for more accurate per-site diversity estimates. We tested SPrUCE on UCE alignments from a range of taxa, including invertebrates and vertebrates (finches, honeybees, sheep and smelt). SPrUCE produces diversity values consistent with whole-genome derived estimates that require an assembled reference. It is fast, scalable, and effective even with missing data. Its modelling approach enables accurate population-level assessments of genetic diversity, offering a new and reliable option for conservation and population genetics.

Abstract Background Extrachromosomal DNA (ecDNA) is a structural variant linked to poor prognosis in pediatric cancers. Patient-derived xenograft (PDX) models are crucial tools for cancer research, as they are believed to recapitulate the molecular features and intratumoral heterogeneity in patient tumors. However, ecDNA demonstrates unique evolutionary dynamics under selective pressure, and its behavior during PDX development remains largely uncharacterized. This study investigates the fidelity of PDX models in representing ecDNA from primary tumors. By analyzing ecDNA sequence composition and copy number conservation across pediatric solid cancers, we assess how well PDX models recapitulate the ecDNA landscape observed in human tumors. Methods AmpliconArchitect was used to analyze whole-genome sequencing (WGS) of 338 PDX models and 127 corresponding primary tumors. ecDNA status, sequence, copy number, and associated genes were compared between PDX models and their matched human tumors. Additionally, multiome RNA and ATAC single-cell sequencing of a PDX tumor enabled comparison of ecDNA intratumoral heterogeneity relative to similar data from the primary tumor. Results ecDNA in PDX models largely recapitulated oncogene amplifications observed in human tumors, with MYCN being the most frequently amplified. ecDNA status remained unchanged for a majority of the PDX models (105/127, 83%) compared to primary tumors, with 20% of previously ecDNA-negative cases acquiring ecDNA during PDX development. Consequently, ecDNA was more prevalent in the PDX models than in their corresponding human tumors (McNemar's test, p = 0.00086). Detailed examination of ecDNA sequences in tumor-PDX pairs showed substantial conservation (67% with > 90% sequence overlap) but variable breakpoint concordance. Single-cell analysis demonstrated that rare ecDNA-positive cells from the primary tumor preferentially drive PDX tumor development. Conclusion This study highlights the prevalence, oncogenic content, and conservation of ecDNA in PDX models relative to pediatric patient tumors. We observed that ecDNA frequently recapitulates oncogene amplifications found in human cancers, is generally preserved during PDX establishment, and reflects subtype-specific patterns across tumor types. These findings support the utility of PDX models in studying ecDNA biology in pediatric cancer progression and therapy. Longitudinal sampling during PDX tumor growth and under therapeutic pressure could provide insights into molecular evolution, clonal selection, and ecDNA-driven therapy resistance.

Motivation Focal oncogene amplification is a key driver of tumor progression. Remarkably, the increased pathology depends on the context-whether the amplification is extrachromosomal (ecDNA) or intrachromosomal. EcDNA amplifications promote heterogeneity, therapy resistance, and poor prognosis. Focal intrachromosomal amplifications often arise through breakage-fusion-bridge (BFB) cycles, which produce highly rearranged but stable chromosomes. Distinguishing BFB from ecDNA remains challenging due to overlapping genomic signatures. To address this, we present BFBArchitect, a computational method leveraging long-read Oxford Nanopore data to identify BFB sequences consistent with both copy number and structural variations.Results We provide a novel combinatorial characterization of BFB, which naturally leads to an integer linear programming (ILP) optimization. The ILP optimization generates a BFB sequence that best explains experimentally observed copy numbers and foldback structural variants. We implement this idea in a tool called BFBArchitect, which achieves near-perfect accuracy in distinguishing BFB from non-BFB structures in extensive simulations as well as on 18 validated tumor samples. Moreover, it generates sequence-level BFB reconstructions that provide mechanistic insights into BFB formation, including repair mechanisms with template switching and other structural variants, and recapture of telomere for stabilization.Availability and implementation BFBArchitect is available at https://github.com/AmpliconSuite/BFBArchitect.

Cancer cells actively release extracellular vesicles (EVs) into the tumor microenvironment, where they interact with both malignant and non-malignant cells, activating signaling pathways and reshaping the microenvironment. In this study, we investigated EVs secreted by FGFR2 -amplified cancers of unknown primary (CUPs), which generate extrachromosomal circular DNA (ecDNA) as a mechanism of oncogene amplification. We found that FGFR2 -containing ecDNA is packaged into both small and large EVs, horizontally transferred to recipient cells, and remains functionally active. Upon exposure to CUP-derived EVs—either by direct administration or co-culture—cancer (NCI-N87, THP1) and non-cancer (HUVEC, fibroblasts) cells internalized FGFR2 ecDNA, which was subsequently transcribed and translated to some extent. Functionally, CUP-derived EVs polarized THP1 cells toward an M2-like phenotype and promoted HUVEC proliferation. In vivo , xenografts generated from CUP cell lines released circulating FGFR2 + EVs, which mediated the systemic transfer of FGFR2 ecDNA to distant organs. Collectively, these findings demonstrate that tumor-derived EVs can propagate and horizontally transfer oncogenic ecDNA both in vitro and in vivo , providing a possible mechanistic basis for the high metastatic potential of this tumor type.

Abstract The amplification of oncogenes on extrachromosomal DNA (ecDNA) enables aggressive, rapidly evolving tumors. Its defiance of Mendelian segregation enables extreme copy number amplifications that escape chromosomal regulatory constraints. Our AmpliconSuite toolset is the most widely used tool for ecDNA analysis in whole genome sequencing data, now deployed on over 43,000 tumor samples. Here, we present novel insights into ecDNA biology enabled through large-scale integrative analysis with these methods. We analyzed 6,366 whole-genome sequenced tumors from combined ICGC and Hartwig Medical Foundation datasets, identifying 2,366 distinct ecDNA capturing >11,000 different genes. Our systematic gene co-amplification analysis revealed striking patterns of gene selection on ecDNA. CDK4-MDM2 co-amplification occurred predominantly via ecDNA (75% of co-amplifications). These loci, normally separated by >11Mbp on chromosome 12, preferentially assembled onto the same ecDNA molecule 93% of the time as revealed by structural analysis of the ecDNA. This suggests selective pressure for maintaining cell cycle and p53 pathway regulators on the same inheritable unit. Our analysis of frequent co-amplifications also revealed that ecDNA preferentially packages chromatin remodelers (NSD3, RSF1) alongside driver oncogenes, as well as including genes that support transcription and translation (INTS4, BRF2), creating self-contained oncogene “support” hubs. Structural analysis revealed cancer type-specific patterns to ecDNA structures, with EGFR ecDNA showing simple architectures in glioblastoma versus complex rearrangements in lung and breast cancers, suggesting distinct formation histories in different cancers. Through AmpliconRepository.org, we provide public access to these ecDNA predictions and co-amplification analysis across major cancer cohorts, currently hosting 16,000+ analyzed samples and 5,000+ characterized ecDNA amplifications. Uniquely open to community contributions, this resource enables researchers to explore patterns across datasets and validate findings. These findings reveal fundamental principles governing ecDNA formation and selection, with implications for understanding tumor heterogeneity, therapeutic resistance, and dependencies which underlie ecDNA-targeted therapies. Citation Format: Jens Luebeck, Ted Liefeld, Edwin Huang, Forrest Kim, Bhargavi Dameracharla, Michael A. Chan, Dhruv Khatri, Kyra Fetter, Kaiyuan Zhu, Thorin Tabor, Soyeon Kim, Hoon Kim, Roel Verhaak, Michael M. Reich, Paul S. Mischel, Jill P. Mesirov, Vineet Bafna. Gene co-amplification and structural patterns reveal principles of extrachromosomal DNA in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 66.

Oncogene amplification on extrachromosomal DNA is a common driver of tumor progression and is associated with acquired drug resistance and poor patient survival. While bulk whole genome sequencing studies have revealed the landscape of genes amplified on extrachromosomal DNA in tumors, it remains challenging to study the subclonal heterogeneity and functional (e.g., transcriptomic) consequences of extrachromosomal DNA on tumors. To address this, we introduce scAmp: a probabilistic algorithm for detecting and analyzing extrachromosomal DNA from single-cell datasets. Using well-characterized cell lines, we demonstrate that scAmp has improved specificity over bulk genome sequencing in predicting extrachromosomal DNA status and can resolve the status of chromosomal amplifications that were historically extrachromosomal. We further showcase scAmp by analyzing 73 patient tumors profiled with single-cell assay for transposase-accessible chromatin by sequencing, where we characterize the subclonal evolution of subclones with extrachromosomal DNA and identify the effect of these amplifications on the chromatin accessibility landscape of cancer cells. Finally, we provide proof-of-concept analyses that scAmp aids in the detection of extrachromosomal DNA from clinical histopathology assays. Together, we anticipate that scAmp will broadly enable further studies – both retrospective and prospective – that dissect critical questions of how extrachromosomal DNAs affect cancer cells and the tumors in which they reside. Oncogene amplification on extrachromosomal DNA (ecDNA) is associated with poor patient prognosis and drug resistance but can be difficult to detect. Here, the authors describe a new computational tool, single-cell amplicon (“scAmp”), which enables the study of ecDNA from single-cell assays.

Oncogene amplification is a key driver of cancer pathogenesis and is often mediated by extrachromosomal DNA (ecDNA). EcDNA amplifications are associated with increased pathogenicity of cancer and poorer outcomes for patients. EcDNA can be detected accurately using fluorescence in situ hybridization (FISH) when cells are arrested in metaphase. However, the majority of cancer cells are non-mitotic and must be analyzed in interphase, where it is difficult to discern extrachromosomal amplifications from chromosomal amplifications. Thus, there is a need for methods that accurately predict oncogene amplification status from interphase cells.We present interSeg, a deep learning-based tool to cytogenetically classify oncogene amplification status as extrachromosomally amplified (EC-amp), intrachromosomally amplified (HSR-amp), or not amplified, from interphase FISH images. We trained and validated interSeg on 652 images (40,446 nuclei). Tests on 215 cultured cell and tissue model images (9,733 nuclei) showed 89% and 97% accuracy at the nuclear and sample levels, respectively. The neuroblastoma patient tissue hold-out set (67 samples and 1,937 nuclei) also revealed 97% accuracy at the sample level in detecting the presence of focal amplification. In experimentally and computationally mixed images, interSeg accurately predicted the level of heterogeneity. The results showcase interSeg as an important method for analyzing oncogene amplifications.

Ecologists and conservation biologists rely on genetic diversity as a key essential biodiversity variable (EBV) used to track population health and dynamics, and utilize the population parameter θ (estimated by the average pairwise genomic distance) as a key metric of diversity. While whole-genome-sequencing (wgs) is increasingly affordable, it will be considerable time before the full diversity of life is represented by high-quality assembled genomes; even then, constant monitoring will still require repeated sampling of populations. In contrast, genome skimming (low-coverage, short-read wgs) is highly cost-effective but challenging to analyze because the coverage is too low for assembly and reliable error correction. Mature methods, such as Mash, exist for estimating pairwise genomic distances based on the Jaccard similarity of k-mer sets computed using sketching techniques. Some, such as Skmer, additionally model the impacts of low coverage. These methods have been successfully applied to assembly-free species identification and phylogenetics; however, their use in population genetics has been limited. This is because these methods implicitly treat genomes as haploid and heterozygosity confounds true estimates of genomic distance for diploid organisms. In this paper, we address this problem through a number of technical advances. First, we use coalescent theory to mathematically derive how the Jaccard index between two diploid samples changes with the scaled population size parameter (θ). Next, we derive an estimator that computes θ from the Jaccard index, in addition to several auxiliary variables, which we also estimate from the genome skims. The resulting method, DipSkmer, enables more accurate estimates of coverage, sequencing error, and pairwise nucleotide distance for diploid samples. Analyses of both simulated and empirical datasets show that for diploids and low distances (e.g., < 2%), DipSkmer produces the most accurate pairwise distance estimates, outperforming existing alignment-free methods such as Mash and Skmer, and closely approximates ANGSD, a reference and alignment-based tool. Availability:The code for DipSkmer is available at https://github.com/echarvel3/ReSkmer/tree/DipSkmer-REFACTOR. Simulation scripts and environments are available at https://github.com/echarvel3/dipskmer_scripts.

Abstract Cancer cells actively release extracellular vesicles (EVs) into the tumor microenvironment, where they interact with both malignant and non-malignant cells, activating signaling pathways and reshaping the microenvironment. In this study, we investigated EVs secreted by FGFR2-amplified cancers of unknown primary (CUPs), which generate extrachromosomal circular DNA (ecDNA) as a mechanism of oncogene amplification. We found that FGFR2-containing ecDNA is packaged into both small and large EVs, horizontally transferred to recipient cells, and remains functionally active. Upon exposure to CUP-derived EVs—either by direct administration or co-culture—cancer (NCI-N87, THP1) and non-cancer (HUVEC, fibroblasts) cells internalized FGFR2 ecDNA, which was subsequently transcribed and translated to some extent. Functionally, CUP-derived EVs polarized THP1 cells toward an M2-like phenotype and promoted HUVEC proliferation. In vivo, xenografts generated from CUP cell lines released circulating FGFR2+ EVs, which mediated the systemic transfer of FGFR2 ecDNA to distant organs. Collectively, these findings demonstrate that tumor-derived EVs can propagate and horizontally transfer oncogenic ecDNA both in vitro and in vivo, providing a possible mechanistic basis for the high metastatic potential of this tumor type. Citation Format: Irene Salamon, Giulia Gallerani, Jens Luebeck, Gianluca Storci, Simone Spandau, Beatrice Fontana, Alessia Soru, Mattia Riefolo, Marco Pagano Mariano, Ilaria Pace, Andrea Cavazzoni, Vineet Bafna, Massimiliano Bonafe', Manuela Ferracin. Horizontal transfer of functional extrachromosomal DNA via extracellular vesicles in FGFR2-amplified cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3345.