Extrachromosomal DNA Research Articles

Abstract 4340: Profiling the evolutionary history of giant cancer cells in sarcomas: Hopeful monsters or an evolutionary dead end

Abstract Background: As tumors evolve, they accumulate somatic mutations that serve as an archeological record of their evolutionary past. Undifferentiated pleomorphic sarcomas (USARCs) are malignant soft tissue tumors that often contain scattered giant cancer cells, or potential ‘hopeful monster cells’, histologically. As USARCs frequently undergo whole genome doubling (WGD) and chromothripsis (Steele et al, 2019), both mutational events are likely to contribute to the formation of giant cancer cells, which frequently display atypical nuclear features such as polyploidy, micronuclei and extrachromosomal DNA. Giant cancer cells are also a common histological finding in other sarcoma types, such as dedifferentiated liposarcomas. Aims: This study aims to leverage single cell sequencing approaches to determine the evolutionary history and genomic landscape of giant cancer cells in sarcomas. Methods: 112 giant cancer cells identified in 8 USARCs, 1 dedifferentiated liposarcoma (DDLPS) and 1 myxofibrosarcoma by histology (i.e. nuclear size and shape, as well as multi-nucleation) were isolated via laser capture microdissection thereby retaining important spatial and morphological information. Once isolated, single cell DNA was extracted and amplified using the Ampli1 whole genome amplification (WGA) kit and underwent WGS. DNA was also estimated at a single cell level in one USARC using FACS. Once sorted into different ploidy populations, single nuclei also underwent WGS. Allele specific copy number aberrations (CNAs) were inferred in all single cells. Bulk tumour and normal WGS was also performed. Results: LCM isolated giant cancer cells exhibited extreme sub-clonal heterogeneity with some demonstrating de novo chromothripsis events that was not present in bulk WGS. FACS analysis and copy number inference of single cancer cells in one USARC also revealed the presence of three ploidy populations, including a haploid, a near diploid and a WGD population. Single cell WGS and copy number profiling demonstrated that following near genome scale haploidisation, multiple haploid cells underwent successive rounds of WGD. Conclusions: Chromothripsis and WGD emerges as major contributors to intra-tumor heterogeneity in sarcoma giant cancer cells. Single cell LCM and WGS revealed that some giant cancer cells contained de novo chromothripsis events that were not observed in bulk WGS. Multiple sub-clonal WGD events also permitted one USARC to continue to explore its fitness landscape contributing to ongoing tumor heterogeneity and lethality. Citation Format: Amy Bowes, Tom Lesluyes, Sara Waise, Annelien Verfaillie, Adrienne Flanagan, Jonas Demeulemeester, Maxime Tarabichi, Nischalan Pillay, Peter Van Loo. Profiling the evolutionary history of giant cancer cells in sarcomas: Hopeful monsters or an evolutionary dead end [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4340.

Abstract 2271: Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts

Abstract Background: Extrachromosomal DNA (ecDNA) is a driver of tumor heterogeneity and is associated with poor survival in many different adult and childhood cancer types. Patient-derived xenograft (PDX) models facilitate preclinical drug testing in an in vivo environment that enables analysis of tumor growth and survival and molecular tumor profiling. These models, combined with single-cell profiling, can be used to study intratumoral heterogeneity under therapeutic pressure and the emergence of drug resistance. Here we investigate the abundance and conservation of ecDNA in PDX models with the aim of assessing the faithful representation of ecDNA compared to the tumors from which they are derived. Methods: Whole-genome sequencing (WGS) was applied to 27 medulloblastoma (MB) PDX models and their tumors of origin using Amplicon Architect, a tool used to reconstruct focally amplified regions of DNA to provide insights into the landscape of cancer genomes. The frequency of ecDNA in these models was also compared to that of ecDNA in the MB patient population. Optical genome mapping enabled accurate and reliable ecDNA sequence reconstructions. Multiome single-cell RNA and ATAC-sequencing of a PDX tumor enabled analysis of ecDNA conservation and intratumoral heterogeneity compared to similar data previously obtained from the primary tumor. Results: Comparative analyses revealed that ecDNA is largely conserved in PDX models, emphasizing their relevance as faithful models of the primary tumors. In addition, we find that ecDNA is significantly more frequent in MB PDX models (16 out of 27 analyzed patient-derived tumors) as compared to their frequency in the overall patient population (~18% of all medulloblastoma tumors contain ecDNA), suggesting ecDNA as a driver for successful establishment of MB PDX models. Single-cell analysis revealed that ecDNA is omnipresent in almost all PDX tumor cells, while its presence was limited to only ~9% of tumor cells in the primary tumor. Conclusion: Analysis of ecDNA in medulloblastoma primary tumors and in PDX models revealed that ecDNA is preserved in PDX models. This is an important characteristic that renders patient-derived mouse xenografts as faithful models of their primary tumors. These results emphasize the relevance of PDX models for preclinical in vivo studies aimed at analyzing the role of ecDNA under therapeutic pressure and for analyzing clonal selection along with molecular evolution of ecDNA during the development of therapeutic resistance. Citation Format: Rishaan Kenkre, Owen Chapman, Jens Luebeck, Vineet Bafna, Robert Wechsler-Reya, Jill Mesirov, Lukas Chavez. Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2271.

Abstract 2012: Extrachromosomal DNA promotes drug resistance in pancreatic ductal adenocarcinoma cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in Western Countries and has been predicted to become the second leading cause of cancer-related deaths by 2030. Difficulties in early detection as well as strong chemoresistance of the disease prevent the successful treatment of PDAC patients. Although we have previously identified CYP3A5 as a mediator of paclitaxel resistance, further targets remain elusive. Thus, we set out to identify these CYP3A5-independent mechanisms which could lead to the identification of novel biomarkers and drug targets to improve PDAC treatment. For this project, pancreatic cancer cell lines were derived from treatment-naive PDAC samples and exposed to increasing paclitaxel concentrations to induce resistance. The cells were characterized on a genomic and transcriptomic level using short-read sequencing as well as optical mapping to characterize potential candidate genes in vitro. In our results, multi-drug resistance protein 1 (ABCB1) was highly upregulated in our differential gene expression analysis and confirmed as a CYP3A5-independent mediator of acquired paclitaxel resistance. Its overexpression was mediated by extrachromosomal DNA (ecDNA) amplifications upon drug treatment. These circular DNAs were characterized by short-read sequencing as well as long-read optical mapping, which revealed the structures of the most abundant molecules. Furthermore, we investigated the biogenesis of the ecDNA. Current models propose different events such as chromothripsis or breakage-fusion-bridge cycles to initiate ecDNA formation, however, knowledge about the underlying mechanisms remains limited. To study the ecDNA dynamics, we generated ecDNA-negative cell clones from a paclitaxel resistant PDAC cell line. Interestingly, our clonal cell population appears to show the capacity for ecDNA de novo generation and quickly adapts to changing selective pressure such as therapy. This finding might unveil new insights into the triggers of ecDNA formation and its dynamic regulation. In conclusion, our results suggest the clinical relevance of ABCB1 as a mediator of paclitaxel resistance in PDAC and we currently validate our findings in patient samples. However, ABCB1 inhibition is associated with severe side effects. Instead, the regulatory pathways associated with enhanced ABCB1 expression and the formation of ecDNAs might provide therapeutic potential. Additionally, the characterization of ecDNA de novo generation might unveil new insights into how cancer cells adapt to changing selective pressures. Citation Format: Tim Vorberg, Manuel Reitberger, Bernardo Rodriguez-Martin, Maja Starostecka, Ornella Kossi, Vanessa Vogel, Jan Korbel, Andreas Trumpp, Martin Sprick. Extrachromosomal DNA promotes drug resistance in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2012.

Abstract 6599: 3D genomic analysis reveals novel enhancer hijacking mechanisms caused by complex structural alterations that drive oncogene overexpression

Abstract Enhancer hijacking, caused by structural alterations on chromosomes as well as extrachromosomal DNA (ecDNA), is a common cancer driver event. The complexity and ubiquity of structural alterations in cancer genomes make it difficult to identify enhancer hijacking with genome sequencing alone. Here we describe a 3D genomics-based analysis called HAPI (Highly Active Promoter Interactions) to characterize enhancer hijacking caused by various types of structural alterations. Applying HAPI analysis to HiChIP data from 34 cancer cell lines, we identified novel enhancers hijacked through chromosomal rearrangements to activate both known and potentially novel oncogenes such as MYC, CCND1, ETV1, CRKL, and ID4, which we validated using CRISPRi assays and RNA-seq analysis. Furthermore, we found that ecDNAs often contain multiple oncogenes from different chromosomes, which cause nested enhancer hijacking among them. For instance, we found that MYC ecDNAs relocate additional oncogenes from other chromosomes such as CDX2, ERBB2, or CD44 near the MYC locus, co-opting MYC’s enhancers for their overexpression, which we validated using dual-color DNA FISH and CRISPRi assays. This multiple oncogenes-involved enhancer hijacking mechanism may suggest novel therapeutic strategies such as targeting either the co-opting oncogenes or the hijacked enhancers for ecDNAs. Our study provides a robust strategy to detect enhancer hijacking events using our publicly available HAPI analysis tool and reveals novel mechanisms underlying oncogene activation caused by chromosomal and extrachromosomal structural alterations. Citation Format: Katelyn L. Mortenson, Courtney Dawes, Emily R. Wilson, Nathan E. Patchen, Hailey Johnson, Jason Gertz, Swneke D. Bailey, Yan Liu, Katherine E. Varley, Xiaoyang Zhang. 3D genomic analysis reveals novel enhancer hijacking mechanisms caused by complex structural alterations that drive oncogene overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6599.

Abstract 5870: Taxane-based chemotherapy leads to acquired resistance through ecDNA-based amplification of multi-drug resistance genes

Abstract Focal amplification on extrachromosomal DNA (ecDNA) drives high gene copy number and intercellular genomic heterogeneity, which can fuel tumor adaptation and drug resistance. Targeted therapies have largely failed in patients with oncogene amplification, and patients whose tumors harbor ecDNA-based gene amplification have particularly worse survival compared to patients whose cancers harbor other genetic drivers. Due to the lack of approved therapies for patients with oncogene amplified cancers, many patients receive chemotherapy; however, resistance often develops through mechanisms such as ATP-binding cassette (ABC) transporter overexpression, rendering cross-resistance to other forms of chemotherapy. We examined whether ecDNA amplification in tumor cell lines drives ABC transporter overexpression to mediate resistance to paclitaxel, a commonly prescribed taxane chemotherapy for cancer patients. We selected non-amplified (DLD1 and SW48) and amplified cell lines, including COLO320DM and COLO320HSR (MYC amplified on ecDNA or chromosomal loci, respectively); SNU16 and H716, (FGFR2 and MYC amplified ecDNA); and H2170 (ERBB2 and MYC amplified on ecDNA). All cell lines developed paclitaxel resistance, with the majority (5/7) gaining focal amplification of ABCB1, which encodes P-glycoprotein (P-gp), a common multi-drug resistance transporter. Four models–COLO320DM, COLO320HSR, SNU16, and H2170—developed ABCB1 amplification on ecDNA; one non-amplified cell line (DLD1) acquired focal ABCB1 amplification via a breakage-fusion-bridge (BFB) mechanism, a distinct form of amplification from ecDNA. ecDNA-based ABCB1 amplifications exhibited hallmarks of ecDNA, including intercellular copy number heterogeneity and paclitaxel dose-dependent rapid evolution of ABCB1 copy number. We next assessed whether drug resistance was reversible in ABCB1 amplified cell lines following removal of paclitaxel. Reversal of resistance was observed in COLO320DM and COLO320HSR via loss of ABCB1 ecDNA within 4-6 weeks. However, in SNU16 and H2170 resistant cells, ABCB1 amplification was retained up to 12 weeks of drug removal, with apparent integration into MYC ecDNA, demonstrating that ecDNA-enabled plasticity can result in both short- and long-term paclitaxel resistance. Although targeted therapies have revolutionized treatment approaches for patients with actionable oncodriver alterations, most cancer patients do not have actionable drivers and most patients still receive chemotherapy at some point in their course of treatment. We demonstrated that ecDNA-based resistance to paclitaxel is common in cancer cell lines and may also drive resistance to chemotherapy in patients. Thus, ecDNA-directed therapies (ecDTx) may be an important treatment option for chemo-resistant patients, who currently do not have many effective therapeutic options. Citation Format: Kristen Holmes, Dean Perusse, Cory DuPai, Homa Hemmati, Julie Weiss, Claire Kuelbs, John Bibay, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Taxane-based chemotherapy leads to acquired resistance through ecDNA-based amplification of multi-drug resistance genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5870.

Abstract 3365: Reliable detection of potentially therapeutically actionable ecDNA using clinical-grade NGS in a large pan-cancer cohort

Abstract Background Focal high-level oncogene amplifications (FH-amp) (e.g., MYC, EGFR) frequently occur on extrachromosomal DNA (ecDNA), highly transcribed units of circular non-chromosomal DNA. FH-amp on ecDNA promote intra-tumoral heterogeneity, resistance to therapies, and poor prognosis. Recently, a Phase 1 clinical trial exploring ecDNA-directed treatments has begun. However, to date, bioinformatic detection of ecDNA has relied on whole-genome sequencing data, and no standard method exists to detect ecDNA on targeted NGS. We thus sought to explore the feasibility of detecting ecDNA in a large cohort of patients with solid tumors sequenced with the targeted NGS panel, MSK-IMPACT, and describe the pan-cancer prevalence of oncogenes amplified on ecDNA. Methods We utilized a novel bioinformatic ecDNA-diagnostic algorithm, ECHO, to determine the presence of oncogene FH-amp on ecDNA (including HSR) vs. chromosomal in clinical-grade NGS data. 72,651 tumor samples profiled by MSK-IMPACT were queried for high level oncogene FH-amp (AR, CCNE1, CDK4/6, EGFR, FGFR1-4, KRAS, MDM2, MDM4, MET, MYC, MYCL, and MYCN) above a fold-change of three, and subsequently annotated with ECHO. Patients with concomitant oncogenic driver mutations or fusions (e.g., EGFR L858R, EML4-ALK) were excluded based on the hypothesis that they might be mutually exclusive with oncogene FH-amp. Results FH-amp of any above listed oncogene were found in 3065 (4.2%) samples. Cancer types with frequent FH-amp without concomitant driver alterations were liposarcoma (LPS) (61% FH-amp; 43% were ecDNA+), glioblastoma (GBM) (22%; 72%), gastroesophageal adenocarcinoma (GE AD) (10%; 57%) squamous cell lung cancer (7%; 18%), and breast cancer (BRCA) (7%; 23%). Among tumors with oncogene FH-amp, GBM and GE AD had high frequencies of ecDNA+ FH-amp of EGFR (39% and 15% of all EGFR ecDNA+). LPS had the highest frequency of ecDNA+ FH-amp of CDK4 (64%), followed by GBM (15%). The majority of ecDNA+ FGFR1 and FGFR2 FH-amp were in BRCA (75% and 47%). EGFR in GBM and EGFR or FGFR2 in GE AD were ecDNA+ in more than 90% of patients, while CDK4 in LPS and FGFR1 in BRCA were ecDNA+ in 45% and 39% respectively.Evaluation the relationship between ecDNA+ by ECHO and amp FC range showed higher ecDNA+ in samples with higher level amp (FC > 5; 58% ecDNA+) compared to lower level amp (FC 4~5; 12%, FC 3~4; 6% ecDNA+). Conclusions A novel ecDNA clinical diagnostic tool, ECHO, was successfully applied to a large clinical cohort of patients enriched for actionable oncogene amplifications sequenced with MSK-IMPACT. The results demonstrate that detection of ecDNA using clinical-grade NGS assays is feasible and that key oncogene FH-amp on ecDNA (e.g., EGFR, FGFR2) are common in select tumor types. ECHO may be developed as a clinical trial device to prospectively identify patients with oncogene ecDNA+ FH-amp for clinical testing of ecDNA-directed therapies. Citation Format: Matteo Repetto, Klaus Wagner, Shalabh Suman, Peter Krein, Pavitra Rao, Maria Arcila, Christian Hassig, Mark Donoghue, Alexander Drilon, Michael Berger. Reliable detection of potentially therapeutically actionable ecDNA using clinical-grade NGS in a large pan-cancer cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3365.

Abstract 2541: The landscape of circular extrachromosomal amplification in pediatric cancers

Abstract Circular extrachromosomal DNA (ecDNA, also known as double minutes, dm) has emerged as a driver of oncogenic copy number amplification, intratumoral heterogeneity, tumor evolution and therapy resistance. We recently screened the whole genomes of 481 medulloblatomas for ecDNA and concluded that medulloblastoma tumors harboring ecDNA are more than twice as likely to progress within 5 years of diagnosis. To evaluate the frequency and clincical impact of ecDNA across the spectrum of childhood cancers, we performed a retrospective analysis of 1671 tumors from 1482 patients across 47 pediatric tumor types from two large cloud pediatric cancer genomics resources, the Children’s Brain Tumor Network and St. Jude Cloud. ecDNA was detected in 15 pediatric tumor types, including ETMR (100%, n=4), osteosarcoma (47%, n=57), rhabdomyosarcoma (40%, n=35), neuroblastoma (30%, n=106), high-grade glioma (20%, n=157), and retinoblastoma (19%, n=32). Patients with tumors containing ecDNA had significantly poorer 5-year overall survival. In sequential samples from the same tumor at diagnosis and relapse, we observe evidence of ecDNA evolution including sequence instability and generation of new high-copy ecDNA amplification not detected at initial diagnosis. These results comprise the most detailed survey to date of the landscape of extrachromosomal amplifications across childhood cancers. Citation Format: Sunita Sridhar, Owen S. Chapman, Shanqing Wang, Aditi Dutta, Jill P. Mesirov, Lukas Chavez. The landscape of circular extrachromosomal amplification in pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2541.

Abstract 4346: Deciphering the landscape of extrachromosomal DNA in gastric cancer and its potential impact on cancer dynamics

Abstract Extrachromosomal DNA (ecDNA) drives substantial copy number increases in focal regions harboring oncogenes and its non-Mendelian inheritance during mitosis contributes to cancer heterogeneity and a poor prognosis across multiple cancer types. However, the prevalence and function of ecDNA in gastric cancer (GC) are still largely unknown. Here, we profiled ecDNA by whole-genome sequencing analysis from 76 GC patients and explored its functional attributes through whole-transcriptome sequencing from the same tissues. Focal somatic copy number amplification (fSCNA) was detected in 27 patients (35.5%), of which 17 (22.4%) had at least one circular form of focal amplification (ecDNA+/fSCNA+). Most of the patients carrying one or more ecDNA were classified as TCGA-CIN subtype (n=12; CIN: chromosomal instability). The fSCNA hotspots were observed at 17q12 and 19q12, where ERBB2 and CCNE1 are located, respectively. Amplifications at both loci occurred in seven patients and were formed into ecDNA in four and two patients, respectively. The ecDNA amplicons carried more oncogenes with significantly higher copy numbers than other amplicons (one-tailed Mann-Whitney U test, p=3.9e-03), and 44.4% (12 of 27) of ecDNA amplicons regions were overlapped with tandem duplication or chromothripsis, known causes of ecDNA formation. Oncogenes such as ERBB2 and MIEN1, residing on ecDNA, exhibited significantly increased expression compared to matched normal tissue as well as patients with the corresponding genes located on their chromosome only, suggesting that the ecDNA-induced copy number increases contribute to elevated expression levels. On the other hand, MYC showed higher expression in the ecDNA+/fSCNA+ group, irrespective of its fSCNA status (padj=0.028 and 4.24e-06 against the ecDNA-/fSCNA+ and ecDNA-/fSCNA- group, respectively). We further explored gene clusters that were associated with the ecDNA+/fSCNA+ group using weighted gene co-expression network (WGCNA) analysis. One module was distinct from ecDNA- patients, consisting of genes associated with the “protein processing in endoplasmic reticulum” and “spliceosome” pathways, inferring that transcription and translation are more active in ecDNA+ patients. Moreover, the overall survival analysis of 76 GC patients revealed that ecDNA+/fSCNA+ patients were significantly associated with poor prognosis (log-rank test, p=0.014). In conclusion, ecDNA may be a main source of high expression of oncogenes that influence GC development, and subsequently upregulated global transcription activity will make GC more aggressive. Citation Format: Donghyeok Seol, Seunghyun Kang, Sanghyun Kim, Jieun Lee, Mira Yoo, Duyeong Hwang, So Hyun Kang, Chanmi Bang, Young Suk Park, Sang-Hoon Ahn, Hyung-Ho Kim, Hoon Kim, Yun-Suhk Suh. Deciphering the landscape of extrachromosomal DNA in gastric cancer and its potential impact on cancer dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4346.

Abstract 6957: Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary

Abstract The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with recipient cells inducing regulation of gene expression, activation of specific signaling pathways and tumor microenvironment remodeling. We investigated the EVs released by two cell lines of cancer of unknown primary site (CUP) that we established from patient’s tumor cells, namely CUP#55 and CUP#96, as described in https://www.medrxiv.org/content/10.1101/2023.03.12.23287041v1). CUP comprises 3-5% of new cancer cases and presents with metastases of unknown or uncertain origin and no apparent primary tumor. Our CUP cell lines are both characterized by FGFR2 gene amplification. It has been recognized that tumors use circular extrachromosomal DNA (ecDNA) as a way to increase oncogenic amplification. We hypothesized that FGFR2 amplification in CUP cell lines could be associated with ecDNA generation and that this ecDNA could be loaded as cargo inside EVs. FGFR2 copy number was quantified using a probe-based droplet digital PCR assay then we identified the different nature of FGFR2 amplification in the two models using FGFR2 FISH assay: CUP#55 presented a chromosomal homogeneously staining region while CUP#96 displayed double minute chromosomes. FGFR2 DNA was detectable in the cytoplasm fraction, therefore we isolated extracellular vesicles from culture medium using an ultracentrifugation-based protocol and confirmed the presence of the entire length of FGFR2 gene inside small and large vesicles. Since ecDNA contributes to cancer genome remodeling also forming chimeric circles, we confirmed the circular nature of a fraction of FGFR2 ecDNA using the Plasmid-Safe ATP-dependent DNAse test. Finally, we tested the oncogene delivery capability of CUP EVs by administering CUP#96 and CUP#55 small and large vesicles to recipient cell lines. We observed an increase of FGFR2 DNA at 24 hours upon EV treatments, and its functional transcription in FGFR2 mRNA after 48 hours. In conclusion, we identified a model of oncogene amplification and delivery in two cell lines of cancer of unknown primary, which could explain the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Citation Format: Irene Salamon, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Giulia Gallerani, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6957.

Abstract 7348: CytoCellDB: A gold standard database for classification and analysis of extrachromosomal DNA in cancer

Abstract Extrachromosomal DNA (ecDNA), or double minute chromosomes, are established cytogenetic markers for malignancy and genome instability. More recently, the cancer community has gained a heightened awareness of the roles of ecDNA in cancer proliferation, drug resistance and epigenetic remodeling. A current hindrance to understanding the biological roles of ecDNA is the lack of available cell line model systems with experimental cytogenetic data that confirm ecDNA status. Although several recent landmark studies have identified numerous cell lines and tumor models with ecDNA, the current sample size limits our ability to detect ecDNA-driven molecular differences due to limitations in power. Increasing the number of model systems that are known to express ecDNA would provide new avenues for understanding the fundamental underpinnings of ecDNA biology and would unlock a wealth of potential targeting strategies for ecDNA-driven cancers. To bridge this gap, we created CytoCellDB, a resource that provides karyotype annotations, including ecDNA status, and leverages publicly available global cell line data from the Cancer Dependency Map (DepMap) and the Cancer Cell Line Encyclopedia (CCLE). We expanded the total number of cancer cell lines with ecDNA annotations to 632, or 34% of the cell lines in CCLE/DepMap. Of these, 154 cell lines express ecDNA, which is a 240% increase from the current sample size. We demonstrate that a strength of our framework is the ability to interrogate ecDNA, and the compendium of other chromosomal aberrations, in the context of cancer-specific vulnerabilities, drug sensitivities, and other multi-omic features (genomics, transcriptomics, methylation, proteomics). We anticipate that this resource will lower the barrier to systematic, population-scale discoveries related to ecDNA and will provide insights into strategies and best practices for determining novel therapeutics that overcome ecDNA-mediated drug resistance. Citation Format: Liz Brunk. CytoCellDB: A gold standard database for classification and analysis of extrachromosomal DNA in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7348.

Abstract 7644: Prognostication of genomic characteristics of residual breast cancer after neoadjuvant chemotherapy

Abstract Residual cancer burden (RCB) based on pathologic characteristics is the most powerful prognostic factor in breast cancer (BC) which received neoadjuvant chemotherapy (NAC) followed by surgery. According to RCB classification, class III predicted short survival duration regardless of BC subtypes according to hormone receptor (HR) and human epidermal growth factor receptor-2 (HER-2) state. However, up to 20-40% of BCs with RCB class III did not experience BC recurrence and had long term survival without disease recurrence. In our study, we evaluated genomic characteristics of BC which had high RCB class. We collected 96 fresh frozen BC specimens which had undergone surgery after NAC between 2010 and 2018 from Samsung Medical Center Biobank. We performed whole genome or exome sequencing, RNA sequencing and high-plex single-cell spatial transcriptomics. Of 96 BCs, 65 BCs had experienced BC recurrence and 31 BCs have not been BC recurrence. Of 65 BCs with dismal prognosis, 12 were HR+HER2-, four in HR+HER2+, 39 in triple negative breast cancer (TNBC) and 10 in HR-HER2+ BC. Of 31 BCs without recurrence, 11 were HR+HER2-, two in HR+HER2+, 14 in TNBC and four in HR-HER2+ BC. In HR+HER2- BC, ten of 12 BCs with dismal prognosis were categorized into basal like intrinsic subtype and two were into luminal B subtype compared that six of luminal A and five of luminal B subtype categorization in good prognosis BC group. Age associated mutation signature were enriched in HR+HER2- BC with good prognosis (p=0.027). Tumor microenvironment (TME) analysis using Kassandra suggested that high monocytes were associated with BC with dismal prognosis (p=0.00031). In single-cell spatial transcriptome analysis, T-cell abundance in the tumoral niche is associated with dismal prognosis in HR+HER2- BC (p=0.039). In TNBC, almost BCs (88.7%) were categorized into basal-like intrinsic subtype regardless of their prognosis. TME analysis presented that immune cells including macrophages and lymphocytes with CD4 and CD8 T cells were significantly enriched in TNBC with good prognosis (ps <0.05, respectively). In HER2+ BC, intrinsic subtypes were heterogeneous in both groups. Focal amplification in HER2+BC patient harbored DNA segments from different chromosomes more frequently than in those with good prognosis (p=0.00091), which was prominent in oncogenic extrachromosomal DNA amplifications compared to other oncogenic amplifications. In TME analysis, there were no specific cell enrichments according to BC prognosis but sing-cell spatial transcriptome analysis revealed that higher T-cell abundance in the tumoral niche was associated with HR+HER2+ BC with good prognosis (p=0.047). In conclusion, each BC subtype had their own genomic characteristics to determine their prognosis. Our study also suggested that TME characteristics was common prognostic factor for survival outcome of BC with residual tumor after NAC. Citation Format: Ji-Yeon Kim, Sabin Park, Sepil Ahn, Eun Seop Seo, Soyeon Kim, Mark Gregory, Emily Killingbeck, Eun Yoon Cho, Jeong Eon Lee, Jin Seok Ahn, Yeon Hee Park, Woong-Yang Park, Hoon Kim, Semin Lee, Young-Hyuck Im. Prognostication of genomic characteristics of residual breast cancer after neoadjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7644.

Abstract 2860: High-resolution mapping of oncogenic structural changes in osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify cancer-specific oncogenic alterations for treatment of osteosarcomas. Osteosarcoma is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. The functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments to affect gene expression. SVs can also lead to the formation of extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers yet they remain poorly understood. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), we studied SVs in osteosarcoma with the goal of elucidating their contribution to cancer development. We used a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies as well as genomically via whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in osteosarcoma, we integrated OGM and WGS data for all 11 cell lines to describe detailed genome-wide SVs including ecDNA formation. PDX lines assayed using OGM exhibited between 106 and 704 translocations each (versus zero detected in germline controls) and we identified dozens of potential ecDNA amplicons, including one amplicon which may explain exceptionally high expression of YAP1 and BIRC3 transcripts. Further, we used HiC to describe the topological environment resulting from these SVs and observed potential enhancer hijacking events affecting dozens of known oncogenes including MYC and CDK4. HiC interaction maps among six cell lines reveal genomic compartments and TAD boundaries in detail, with 4,167 unique TAD boundaries identified among six cell lines using 50kb genomic bins. Despite the observed complexity of these genomes, we note that 71% of TAD boundaries are consistent in at least four of the six HiC assayed cell lines, and 44% are present in all six lines. Changing TAD boundaries are associated with numerous SVs and/or copy number discrepancies, and are associated with genomic regions that contain genes with dysregulated RNA transcript levels. Overall, OGM and HiC modalities add considerably to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew Scott Clugston, Stan Leung, Eunice Fuentes, Leanne Sayles, Megan Ostrowski, Coco Wu, Christina Curtis, Zhicheng Ma, Yanding Zhao, Georgi K. Marinov, Vijay Ramani, Marcus Breese, Alejandro Sweet-Cordero. High-resolution mapping of oncogenic structural changes in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2860.

Abstract 604: Targeting casein kinase 1 alpha (CK1 alpha) and transcriptional CDKs (CDK7/9) in human liposarcomas

Abstract Introduction: Liposarcomas (LPS) are rare mesenchymal cell malignancies of adipocytic origin that are diagnosed in more than 3500 patients in the US each year. The two most common subtypes, well-differentiated LPS (WDLPS) and dedifferentiated LPS (DDLPS), are characterized by extrachromosomal DNA amplifications harboring the MDM2 (100%) and CDK4 (90+%) genes, generally with wild type TP53. Management of metastatic or surgically unresectable LPS remains purely palliative. Recent clinical trials targeting MDM2 or CDK4/6 with small-molecule inhibitors have shown promise but have been hampered by dose-limiting toxicities. The development of new therapeutics is greatly needed to improve outcomes for patients with LPS. Results: To identify unique liposarcoma-specific vulnerabilities, we screened multiple human LPS cell lines for transcriptional CDK expression and found high levels of CDK7 and CDK9. We demonstrate that CDK9 inhibitors suppress LPS cell growth and induce apoptosis by decreasing MDM2 levels while inducing expression of p53. To enhance p53 activation in these cells, we screened for expression of known regulators of p53, including CK1α, whose inhibition has previously been shown to activate p53. We demonstrate that LPS cells express CK1α and that the cytotoxic effects of CDK9 inhibitors are enhanced upon CK1α depletion. These data led us to examine combined targeting of CK1α and CDK9 in LPS with the novel agent BTX-A51, which has previously been shown to inhibit CK1α, CDK9, as well as CDK7 with nanomolar efficacy in AML models. BTX-A51 potently reduces expression of MDM2 with marked induction of p53, resulting in profound apoptosis of LPS cells. Through CRISPR/Cas9-mediated p53 knockout, we establish that BTX-A51-mediated apoptosis is primarily p53-dependent. However, BTX-A51 also reduces expression of MCL1 and primes LPS cell lines and primary LPS cells for BIM-induced apoptosis, as demonstrated by BH3 profiling. Importantly, preliminary in vivo data in an LPS patient-derived xenograft model reveal that BTX-A51 is well-tolerated with tumor growth inhibition. Conclusions: Our results suggest that BTX-A51 has potent preclinical efficacy in treating LPS, primarily through inhibition of CK1α and CDK9. Future mechanistic studies will further clarify mechanisms of BTX-A51-mediated apoptosis, as well as the contribution of CDK7 inhibition to anti-tumor activity. Our data justify a planned clinical trial that will evaluate the efficacy of BTX-A51 in patients with advanced WDLPS or DDLPS. Citation Format: Renyan Liu, Nicole L. Solimini, Patrick Bhola, Timothy B. Branigan, Jie Hao, Xin Wang, Roshen Alharthi, Michael Yorsz, Shaili Soni, Cing-siang Hu, Irit Snir-Alkalay, Prafulla Gokhale, Anthony Letai, Yinon Yinon Ben-Neriah, George D. Demetri, Geoffrey I. Shapiro. Targeting casein kinase 1 alpha (CK1 alpha) and transcriptional CDKs (CDK7/9) in human liposarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 604.

Abstract 3887: The origins and clinical impact of extrachromosomal DNA across 39 cancers

Abstract Extrachromosomal DNA (ecDNA) is emerging as a major contributor to cancer drug resistance and poor outcome in many cancer types. To understand the underlying genetic and mutational processes associated with ecDNA formation and presence, we conducted an analysis of whole-genome sequencing data from 14,778 cancer patients representing 39 tumor types from the Genomics England cohort. Our findings revealed focal amplifications driven by ecDNA in 17.1% of tumor samples, exhibiting varying frequencies across tissues. Notably, liposarcomas (55.4%) and HER2+ breast cancers (46.4%) displayed higher rates compared to lower rates found in pancreatic adenocarcinomas (2%). In addition to oncogenic amplifications such as HER2 derived from ecDNA during cancer evolution, ecDNAs that did not contain oncogenes were enriched for immunomodulatory genes (e.g., CXCL6, LRRC32, and HLA-G). Immunomodulatory ecDNAs exhibited reduced T cell infiltration. In the context of intrinsic and environmental mutational processes, deamination clock-like, tobacco smoking, and APOBEC cytidine deaminase signatures positively correlated with ecDNA presence. Conversely, mismatch repair deficiency (MMRd) and POLD1/POLE deficiency signatures exhibited a negative correlation. Mutational processes associated with tobacco exposure, UV light, and spontaneous deamination primarily occurred before ecDNA formation, while signatures of homologous recombination repair deficiency tended to manifest post-ecDNA formation.Notably, ecDNA was enriched at metastatic sites and significantly associated with shorter overall survival in multivariable analysis (HR 1.31, 95% CI 1.17-1.47), underscoring its clinical relevance. This study enhances our understanding of the mutational processes and tissue contexts that shape the ecDNA landscape in human cancer and highlights its implications for clinical outcomes. Citation Format: Chris Bailey, Oriol Pich, Kerstin Thol, Jens Luebeck, Georgia Stavrou, Thomas B. Watkins, Natasha E. Weiser, Wei-Ting Lu, Jeanette Kittel, Robert Bentham, Natasha Sharma, Roberto Salgado, Dave Moore, Maria Litovchenko, King L. Hung, Alex J. Cornish, Bhargavi Dameracharla, Serena Nik-Zainal, Vineet Bafna, Howard Chang, Nicholas McGranahan, Nnennaya Kanu, Genomics England Consortium, Paul S. Mischel, Mariam Jamal-Hanjani, Charles Swanton. The origins and clinical impact of extrachromosomal DNA across 39 cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3887.

Identification and characterization of extrachromosomal circular DNA in large-artery atherosclerotic stroke.

Extrachromosomal circular DNA (eccDNA) is a new biomarker and regulator of diseases. However, the role of eccDNAs in large-artery atherosclerotic (LAA) stroke remains unclear. Through high-throughput circle-sequencing technique, the length distribution, genomic characteristic and motifs feature of plasma eccDNA from healthy controls (CON) and patients with LAA stroke were analysed. Then, the potential functions of the annotated eccDNAs were investigated using GO and KEGG pathway analyses. EccDNAs mapped to the reference genome showed SHN3 and BCL6 were LAA stroke unique transcription factors. The genes of differentially expressed eccDNAs between LAA stroke patients and CON were mainly involved in axon/dendrite/neuron projection development and maintenance of cellular structure via Wnt, Rap1 and MAPK pathways. Moreover, LAA stroke unique eccDNA genes played a role in regulation of coagulation and fibrinolysis, and there were five LAA stroke unique eccDNAs (Chr2:12724406-12724784, Chr4:1867120-186272046, Chr4:186271494-186271696, Chr7:116560296-116560685 and Chr11:57611780-5761192). Additionally, POLR2C and AURKA carried by ecDNAs (eccDNA size >100 kb) of LAA stroke patients were significantly associated with development of LAA stroke. Our data firstly revealed the characteristics of eccDNA in LAA stroke and the functions of LAA stroke unique eccDNAs and eccDNA genes, suggesting eccDNA is a novel biomarker and mechanism of LAA stroke.

Dynamics of extrachromosomal circular DNA in rice

The genome’s dynamic nature, exemplified by elements like extrachromosomal circular DNA (eccDNA), is crucial for biodiversity and adaptation. Yet, the role of eccDNA in plants, particularly rice, remains underexplored. Here, we identify 25,598 eccDNAs, unveiling the widespread presence of eccDNA across six rice tissues and revealing its formation as a universal and random process. Interestingly, we discover that direct repeats play a pivotal role in eccDNA formation, pointing to a unique origin mechanism. Despite eccDNA’s prevalence in coding sequences, its impact on gene expression is minimal, implying its roles beyond gene regulation. We also observe the association between eccDNA’s formation and minor chromosomal deletions, providing insights of its possible function in regulating genome stability. Further, we discover eccDNA specifically accumulated in rice leaves, which may be associated with DNA damage caused by environmental stressors like intense light. In summary, our research advances understanding of eccDNA’s role in the genomic architecture and offers valuable insights for rice cultivation and breeding.

Enhancer infestation drives tumorigenic activation of inactive B compartment in Epstein-Barr virus-positive nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignant epithelial tumor endemic to Southern China and Southeast Asia. While previous studies have revealed a low frequency of gene mutations in NPC, its epigenomic aberrations are not fully elucidated apart from DNA hypermethylation. Epigenomic rewiring and enhancer dysregulation, such as enhancer hijacking due to genomic structural changes or extrachromosomal DNA, drive cancer progression. We conducted Hi-C, 4C-seq, ChIP-seq, and RNA-seq analyses to comprehensively elucidate the epigenome and interactome of NPC using C666-1 EBV(+)-NPC cell lines, NP69T immortalized nasopharyngeal epithelial cells, clinical NPC biopsy samples, and invitro EBV infection in HK1 and NPC-TW01 EBV(-) cell lines. In C666-1, the EBV genome significantly interacted with inactive B compartments of host cells; the significant association of EBV-interacting regions (EBVIRs) with B compartment was confirmed using clinical NPC and invitro EBV infection model. EBVIRs in C666-1 showed significantly higher levels of active histone modifications compared with NP69T. Aberrant activation of EBVIRs after EBV infection was validated using invitro EBV infection models. Within the EBVIR-overlapping topologically associating domains, 14 H3K4me3(+) genes were significantly upregulated in C666-1. Target genes of EBVIRs including PLA2G4A, PTGS2 and CITED2, interacted with the enhancers activated in EBVIRs and were highly expressed in NPC, and their knockdown significantly reduced cell proliferation. The EBV genome contributes to NPC tumorigenesis through "enhancer infestation" by interacting with the inactive B compartments of the host genome and aberrantly activating enhancers. The funds are listed in the Acknowledgements section.

Barley AGO4 proteins show overlapping functionality with distinct small RNA-binding properties in heterologous complementation

Key messageBarley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5′ terminal nucleotide of small RNAs, demonstrating functional conservation and divergence.The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5′ adenine residue, while also accepting 5′ guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5′ adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation.

MUC20 regulated by extrachromosomal circular DNA attenuates proteasome inhibitor resistance of multiple myeloma by modulating cuproptosis

BackgroundProteasome inhibitors (PIs) are one of the most important classes of drugs for the treatment of multiple myeloma (MM). However, almost all patients with MM develop PI resistance, resulting in therapeutic failure. Therefore, the mechanisms underlying PI resistance in MM require further investigation.MethodsWe used several MM cell lines to establish PI-resistant MM cell lines. We performed RNA microarray and EccDNA-seq in MM cell lines and collected human primary MM samples to explore gene profiles. We evaluated the effect of MUC20 on cuproptosis of PI-resistant MM cells using Co-immunoprecipitation (Co-IP), Seahorse bioenergetic profiling and in vivo assay.ResultsThis study revealed that the downregulation of Mucin 20 (MUC20) could predict PI sensitivity and outcomes in MM patients. Besides, MUC20 attenuated PI resistance in MM cells by inducing cuproptosis via the inhibition of cyclin-dependent kinase inhibitor 2 A expression (CDKN2A), which was achieved by hindering MET proto-oncogene, receptor tyrosine kinase (MET) activation. Moreover, MUC20 suppressed MET activation by repressing insulin-like growth factor receptor-1 (IGF-1R) lactylation in PI-resistant MM cells. This study is the first to perform extrachromosomal circular DNA (eccDNA) sequencing for MM, and it revealed that eccDNA induced PI resistance by amplifying kinesin family member 3 C (KIF3C) to reduce MUC20 expression in MM.ConclusionOur findings indicated that MUC20 regulated by eccDNA alleviates PI resistance of MM by modulating cuproptosis, which would provide novel strategies for the treatment of PI-resistant MM.

Novel molecular subtypes of intracranial germ cell tumors expand therapeutic opportunities.

Intracranial germ cell tumors (IGCTs) are a rare group of malignancies that are clinically classified as germinomas and nongerminomatous germ cell tumors (NGGCTs). Previous studies have found that somatic mutations involving the mitogen-activated protein kinase/mTOR signaling pathway are common early events. However, a comprehensive genomic understanding of IGCTs is still lacking. We established a cohort including over 100 IGCTs and conducted genomic and transcriptomic sequencing. We identified novel recurrent driver genomic aberrations, including USP28 truncation mutations and high-level copy number amplification of KRAS and CRKL caused by replication of extrachromosomal DNA. Three distinct subtypes associated with unique genomic and clinical profiles were identified with transcriptome analysis: Immune-hot, MYC/E2F, and SHH. Both immune-hot and MYC/E2F were predominantly identified in germinomas and shared similar mutations involving the RAS/MAPK signaling pathway. However, the immune-hot group showed an older disease onset age and a significant immune response. MYC/E2F was characterized by a younger disease onset age and increased genomic instability, with a higher proportion of tumors showing whole-genome doubling. Additionally, the SHH subtype was mostly identified in NGGCTs. Novel genomic aberrations and molecular subtypes were identified in IGCTs. These findings provide molecular basis for the potential introduction of new treatment strategies in this setting.