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Extrachromosomal DNA circularization is a common event in cancer cells and frequently serves as a vehicle for cancer oncogene amplification. Random segregation of oncogene-containing extrachromosomal circular DNA promotes rapid intercellular heterogeneity, conferring tumors the ability to rapidly evolve and escape therapy. Smaller, copy-number neutral extrachromosomal circular DNAs are also abundantly identified in both healthy and malignant tissues, but their function in cancer is still unknown. Understanding how extrachromosomal circular DNAs contribute to intercellular heterogeneity in cancer cells remains crucial, however methods for an unbiased characterization of extrachromosomal circular DNAs in single cells are lacking. We introduce scEC&T-seq (single cell extrachromosomal circular DNA and transcriptomic sequencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cells. We demonstrate the ability of our method to isolate and detect extrachromosomal circular DNAs genome-wide from all range of sizes in single cells. We observed that whereas large oncogene-containing circular DNAs are clonally present in most cancer cells, only a very small fraction of small circular DNAs are recurrently identified in single cells, indicating yet unknown prerequisites for maintenance and propagation. Our method was able to capture and recapitulate the structural complexity of oncogene-containing extrachromosomal circular DNAs in single cells, and the matching transcriptomic data allowed us to identify fusion transcripts resulting from the rearranged extrachromosomal structures. In addition, we observed that whereas the main structure of extrachromosomal circular DNAs is mostly stable in single cells, intercellular differences in extrachromosomal circular DNAs’ content can drive differences in oncogene transcription levels in single cells. We envision that by integrating extrachromosomal circular DNA and mRNA sequencing, our method will not only be useful to investigate the impact of intercellular heterogeneity in extrachromosomal circular DNA in tumor evolution, but also to interrogate its function in other biological and pathological processes. Citation Format: Rocio Chamorro Gonzalez, Thomas Conrad, Robin Xu, Madalina Giurgiu, Maja Cwikla, Katharina Kasack, Lotte Brückner, Eric van Leen, Elias Rodriguez-Fos, Konstantin Helmsauer, Heathcliff Dorado Garcia, Yi Bei, Karin Schmelz, Sascha Sauer, Angelika Eggert, Johannes H. Schulte, Roland F. Schwarz, Kerstin Haase, Richard P. Koche, Anton G. Henssen. Dissecting intercellular extrachromosomal circular DNA heterogeneity in single cancer cells with scEC&T-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1693.

Introduction: Highly variable clinical outcomes including immediate fatal progression to spontaneous regression are characteristically observed for neuroblastoma (NB), a pediatric cancer arising from progenitors of the sympathetic nervous system. Pronounced rates of relapse and fast-evolving resistance against standard-of-care (SOC) treatments, together with a high degree of mutational mobility and heterogeneity, indicate the need for more precise and molecular targeted strategies. Aim: Patient-derived xenograft (PDX) models closely resemble human tumor biology from primary tissue and could be utilized for predictive preclinical testing. For this aim we established a cohort of 10 NB PDX models from 33 primary human tissue samples obtained from the Charité University Clinic. Specimen originated from proliferating areas while possible necrotic lesions had been removed prior to subcutaneous transplantation to immunodeficient NOG mice. After documenting stable growth over 3-4 initial passages, NB PDX models were screened for sensitivity to SOC drugs including actinomycin D, dacarbazine, docetaxel, doxorubicin, etoposide, ifosfamide, gemcitabine, and vincristine. Results: Characteristic NB pathology of all PDX was verified using HE and IHC staining of FFPE tumor sections comparing individual passages. Post-transplant lymphoproliferative disorder (PTLD) was excluded analyzing hCD45 specific marker. Depending on traceable somatic mutations, NB PDX doubling times dramatically varied between 4 days in aggressive, 8 days in intermediate, and up to 18 days in slowly growing tumors. Not surprisingly, high-risk NB tumors with confirmed MYCN amplification and ALK mutation exhibited the fastest growth in our cohort. In contrast, doubling rates of tumors with single ALK mutation or TERT rearrangement were not significantly different compared to the slowest PDX models without driver mutations. Most effective treatment response was observed for vincristine, leading to stable disease or partial regression in 5 out of 8 models (63%) achieving a tumor growth inhibition (TGI) of 70%, whereas all models were insensitive against dacarbazine treatment with a TGI of 33%, respectively. Conclusion: Currently, whole-exome sequencing of all models is performed. Molecular analysis will enable prediction of more precise therapeutic strategies. We will use our NB PDX to validate targeted treatment effects of recent pipeline drugs. As part of the European Innovative Therapies for Children with Cancer-Paediatric Preclinical Proof of Concept Platform (ITCC-P4) consortium, these findings will be correlated with predictions of the NB PDX molecular profile to achieve valuable benefit for pediatric patients. Citation Format: Dennis Gürgen, Jana Rolff, Johannes H. Schulte, Hedwig E. Deubzer, Karin Schmelz, Anton G. Henssen, Patrick Hundsdörfer, Georg Seifert, Angelika Eggert, Wolfgang Walther, Jens Hoffmann. Patient-derived xenograft models of neuroblastoma as improvement for the prediction of targeted therapies for childhood cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A02.