{"paper_id":"945dd0ca-f47c-4955-af91-30860229687d","body_text":"The distribution of the extrachromosomal DNA molecules in early lung cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The distribution of the extrachromosomal DNA molecules in early lung cancer Jianfei Fang, Lisha Ying, Zhengxiao Ma, Ying Yang, Rui Zhu, Dan Su This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4010987/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Lung cancer (LC) is a cancer with high mortality worldwide. Research on the distribution and nature of extrachromosomal DNA molecules (EcDNAm) in early LC is scarce. Methods After removing linear DNA and mitochondrial circular DNA, EcDNAm were extracted from two paired LC tissue samples and amplified using rolling circle amplification. High throughput extrachromosomal DNA or RNA sequencing and bioinformatics analysis were used to explore the distribution and nature of the EcDNAm. To learn more about the role of oncogenes with large EcDNAm sizes, gene onology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Results RNA sequencing results revealed the significant difference in some genes between tumor and corresponding normal samples. At the same time, obvious distinctions were observed between relapsed and non-relapsed tumor samples. The nature of the EcDNAm was comparable between LC samples and matched normal samples. Compared with the matched normal samples, the number of EcDNAm with longer size (EcDNA), which contained driver oncogenes, was relatively high. The majority of EcDNA in this study was mainly focused on the tumor samples. Enrichment analysis of the cancer samples revealed enrichment in biological processes, such as positive regulation of protein localization, axon development and in utero embryonic development. Conclusions This study demonstrated the universality of the distribution and described the nature of EcDNAm in early LC. Moreover, our work fills the investigation of the EcDNAm gap and future studies should focus on the application of EcDNA as a potential biomarker in patients with early LC. extrachromosomal DNA molecules (EcDNAm) lung cancer(LC) high throughput sequencing EcDNA bioinformatics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Lung cancer (LC) has the highest morbidity and mortality rate in the world ( 1 , 2 ). Non-small cell lung cancer (NSCLC) accounts for three quarters of LC ( 3 ). With the popularization of CT examination and low-dose spiral CT screening for LC, the number of patients with early NSCLC is increasing year by year ( 4 ). Compared to targeted therapy and immunotherapy for patients with advanced lung cancer, surgical therapy is the main treatment for patients with early LC, but the prognosis is different ( 5 – 7 ). According to the eighth edition of the staging classification, the 2-year recurrence and metastasis rate of surgically resected stage I NSCLC patients are 38%, and the 5-year overall survival rate is 60–74% ( 8 , 9 ). To improve the survival rate of patients with early NSCLC, it is particularly important to find potential biomolecular targets and to elucidate their roles and mechanisms in the process of recurrence and metastasis. Apart from 22 linear autosome pairs and a pair of sex chromosomes, extrachromosomal DNA molecules (EcDNAm) were found in the human genome ( 10 , 11 ). With the explosive growth of next generation sequencing (NGS) biological data in recent years, EcDNAm has become a research hotspot in the field of biomedical oncology ( 12 – 15 ). EcDNAm were found to be a closed loop structure (16, 17). Wu e t al. reported that EcDNAm with longer sizes are often enriched in the process of tumor formation and aging, and participate in the occurrence and development of tumor aging in a special way ( 18 – 20 ). At the same time, the chromatin of EcDNAm with longer sizes is in a highly open state, in which the transcription of genes is abnormally active, and the mutation of the oncogenic driver gene mediates drug resistance ( 19 , 21 ). These findings indicated that EcDNAm plays an important role in tumorigenesis. However, the mechanism of action of EcDNAm in patients with early stage LC remains unclear. In this study, we explored the mechanism of action of EcDNAm in early stage of LC. To the best of our knowledge, this is the first report to investigate the distribution, function, and mechanism of EcDNAm in matched early LC samples. Materials and methods Tissue DNA preparation and extrachromosomal DNA molecules sequencing The study protocol was approved by the institutional review board at Zhejiang Cancer Hospital (IRB-2020-63). Written informed consent was obtained from two patients with early lung cancer at the Department of Thoracic Surgery at Zhejiang Cancer Hospital. Two tumor tissue samples and matched non-tumor lung cancer samples were collected during the surgery. High-throughput extrachromosomal DNA sequencing was performed by Novogene Biotech Inc. (Beijing, China). Extrachromosomal DNA purification and sequencing were performed according to a previously publication method( 16 ). Sequencing analysis of extrachromosomal DNA molecules Sequencing data were obtained using an Illumina NovaSeq 600 sequencer in 150bp paired-end mode. After that, the raw sequencing data were further quality controlled; for instance, low-quality reads were discarded, and the adaptor was removed. Some bioinformatics tools, such as circle-map, samtools and bedtools, have been used to detect extrachromosomal DNA molecules. Gene function and pathway analysis were performed using the GSEA package. Statistical analysis All statistical analyses and diagram drawings were processed by the R software (version 4.1.2, R Foundation for Statistical Computing, Vienna, Austria). DEseq2 software was used to analyze the expression of 59 early lung cancer patients and their corresponding normal samples using RNA transcriptome data. P < 0.05 was considered for the statistical significance. Results Differential Expression between tumor and corresponding normal samples using RNA sequencing data To investigate the expression level of genes on linear chromosomes in patients with early lung cancer, we used transcriptome data (available from the published article: PMID = 37694148) to analyze the expression of 59 early lung cancer patients and corresponding normal samples. As shown in Fig. 1 , we found a significant difference between the tumor and corresponding normal samples for some genes, such as EGFR. At the same time, obvious distinctions were observed between relapsed and non-relapsed tumor samples. Genome-wide detection of EcDNAm in paired LC samples EcDNAm were detected in 23 pairs of chromosomes by mapping the clean reads to the genome. As showed in Fig. 2 , these results indicate that the presence of EcDNAm is common in tumors and matched normal samples. The genomic distribution of EcDNAm revealed that it was common in all 23 pairs of chromosomes. Few EcDNAm were detected in the mitochondria. According to the previous classification criteria ( 22 ), EcDNAm is generally divided into two types. One is extrachromosomal circular DNA ( EccDNA) , which is usually less than 1KB and cannot be seen under an optical microscope. EccDNA mainly includes telomere rings, small polydisperse DNA elements and micro-DNA. The other is extrachromosomal DNA ( EcDNA) , which is usually larger than 1 MB. EcDNA carries one or more oncogenic driver genes, and EcDNA is visible by light microscopy ( 22 ). As shown In Fig. 2 , the majority of the EcDNA was mainly focused on the tumor samples. Comparing the discrepancy between LC and matched normal samples in EcDNA Most of the large EcDNA molecules (>1 MB) were detected between the LC and matched normal samples. As showed in Fig. 3 , the number of genes contained in EcDNA molecules varied in chromosome between LC and matched normal samples. Previous studies have demonstrated that large EcDNA molecules carried one or multiple driver oncogenes ( 18 , 22 , 23 ). Thus, the distribution of driver oncogenes derived from EcDNA molecules was examined. Compared to the matched normal samples, the number of EcDNA molecules that contained driver oncogenes was relatively high (Fig. 4 ). GO and KEGG pathway analysis of genes in EcDNA molecules GO analysis was performed to examine the functions of genes associated with EcDNA molecules, such as related cellular components (CC), molecular functions (MF), and biological processes (Table 1 ). The dominant biological processes were related to the positive regulation of protein localization, axon development and in utero embryonic development in the tumor samples. The detailed results of gene function are shown in Fig. 5 . Table 1 Go analysis of the genes associated with EcDNA in paired early LC samples Sample Category Description Case 1 BP positive regulation of protein localization response to xenobiotic stimulus small GTPase mediated signal transduction axonogenesis axon development mononuclear cell differentiation embryonic organ development regulation of neuron projection development in utero embryonic development small molecule catabolic process CC glutamatergic synapse neuronal cell body mitochondrial matrix cell-substrate junction focal adhesion collagen-containing extracellular matrix synaptic membrane neuron to neuron synapse cell leading edge postsynaptic specialization MF actin binding protein serine/threonine kinase activity Normal 1 BP small molecule catabolic process regulation of anatomical structure size regulation of cellular component size small GTPase mediated signal transduction axonogenesis embryonic organ development response to nutrient levels mitotic cell cycle phase transition regulation of immune effector process regulation of trans-synaptic signaling CC glutamatergic synapse mitochondrial matrix neuronal cell body postsynaptic specialization asymmetric synapse cell leading edge postsynaptic density coated vesicle neuron to neuron synapse nuclear speck Case 2 BP response to xenobiotic stimulus positive regulation of protein localization embryonic organ development in utero embryonic development axon development axonogenesis regulation of anatomical structure size double-strand break repair small GTPase mediated signal transduction response to nutrient levels CC glutamatergic synapse cell-substrate junction focal adhesion synaptic membrane cell leading edge neuronal cell body collagen-containing extracellular matrix neuron to neuron synapse postsynaptic membrane vesicle lumen MF actin binding Normal 2 BP mononuclear cell differentiation lymphocyte differentiation response to xenobiotic stimulus axonogenesis embryonic organ development response to nutrient levels cell fate commitment epidermis development cytokine-mediated signaling pathway small GTPase mediated signal transduction CC glutamatergic synapse cell-substrate junction neuron to neuron synapse focal adhesion postsynaptic specialization mitochondrial matrix asymmetric synapse synaptic membrane postsynaptic density collagen-containing extracellular matrix MF protein serine/threonine kinase activity actin binding DNA-binding transcription factor binding protein serine kinase activity RNA polymerase II-specific DNA-binding transcription factor binding BP, Biological Process; CC, Cellular Component; MF, Molecular Function In addition, KEGG pathway analysis indicated that the main pathway was involved in the cell cycle which departed from normal samples (Fig. 6 ). Discussion An extrachromosomal DNA molecule (EcDNAm) is circular DNA that plays an important role in the development and heterogeneity of cancer ( 13 , 24 , 25 ). However, the structure, composition and genome-wide frequency of extrachromosomal DNA have not been extensively profiled. In this study, high-throughput sequencing demonstrated the presence of EcDNAm in paired early LC samples, in agreement with previous studies of other cancer types ( 26 , 27 ). In contrast, our data demonstrated that EcDNAm could be found in human genome, with the exception of mitochondria, in early lung cancer. Based on size, two classes of EcDNAm exist in human cells: small (EccDNA) and large (EcDNA). Previous studies have shown that oncogenes in EcDNA are highly expressed ( 19 , 28 ). Compared with the matched normal samples, the number of EcDNAm with a longer size (namely EcDNA), which contained driver oncogenes (for instance, EGFR) was relatively high. Furthermore, the majority of EcDNA was mainly focused on tumor sample, and no obvious differences were found between LC and matched normal samples in this study. GO analysis of genes associated with EcDNA showed that the dominant biological processes were related to positive regulation of protein localization, axon development and in utero embryonic development in tumor samples. KEGG pathway analysis of genes associated with EcDNA indicated that the main pathway was involved in the cell cycle, which departed from normal samples. In this study, we found a significant difference between tumor and corresponding normal samples for some genes, such as EGFR. Based on the limited data and experimental validation, the carcinogenic mechanism of the relationship between chromosomal DNA and extra-chromosomal DNA remains unclear. In the future, we will increase the sample size and further explore the functional mechanism of EcDNAm in early LC. We believe that our findings fill the investigation of the EcDNAm gap in early LC, and that future studies should focus on the application of EcDNA as a potential biomarker and therapeutic target in patients with early LC. Conclusions In conclusion, our study confirmed the genome-wide presence of EcDNAm in parired LC samples. In addition, our work revealed the potential mechanisms of EcDNAm in early LC. This work provides further insights into our understanding of genome plasticity and the role of EcDNAm in early LC, and may contribute to the development of potential clinical therapies. Declarations Author’s contributions Jianfei Fang and Dan Su conceived, designed, and revised the study. Jianfei Fang analyzed the data and wrote the manuscript. Lisha Ying, Zhengxiao Ma, Ying Yang and Rui Zhu analyzed the data. All authors have read and approved the final manuscript. Ethical approval and consent to participate This study protocol was approved by the institutional review board at Zhejiang Cancer Hospital (IRB-2020-63). Written informed consent was obtained from two patients with early lung cancer at the Department of Thoracic Surgery at Zhejiang Cancer Hospital. This study was performed in line with the principles of the Declaration of Helsinki. Acknowledgments This study was supported by the Natural Science Foundation of Zhejiang Province, China (No.LQ21F010001). Funding This study was supported by the Natural Science Foundation of Zhejiang Province, China (No.LQ21F010001). Availability of data and materials The datasets generated during and/or analyzed during the current study are available in the GSA for Human (https://ngdc.cncb.ac.cn.gsa-huaman/), with access numbers PRJCA010105. The extrachromosomal sequencing datasets are not publicly available but are available from the corresponding author upon reasonable request. Consent for publication Not application Competing interests The authors declare no competing interests Author details 1 Department of Pathology, Zhejiang Cancer Hospital, Hangzhou Institute of Medicine(HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China 2 Zhejiang Cancer Institute, Zhejiang Cancer Hospital, Hangzhou Institute of Medicine(HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China 3 Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital) , Hangzhou, Zhejiang, 310022, China. 4 The Second Clinical Medical College, Zhejiang Chinese Medicine University, Hangzhou, Zhejiang 310053, China References Teixeira VH, Pipinikas CP, Pennycuick A, Lee-Six H, Chandrasekharan D, Beane J, et al. Deciphering the genomic, epigenomic, and transcriptomic landscapes of pre-invasive lung cancer lesions. Nature medicine. 2019;25(3):517-25. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics in China, 2015. 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Extrachromosomal circular DNA and structural variants highlight genome instability in Arabidopsis epigenetic mutants. Nature communications. 2023;14(1):5236. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4010987\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":276390135,\"identity\":\"d6a4219e-2dc6-4d60-8226-8b0917657906\",\"order_by\":0,\"name\":\"Jianfei Fang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhejiang Cancer Hospital, Chinese Academy of Sciences\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jianfei\",\"middleName\":\"\",\"lastName\":\"Fang\",\"suffix\":\"\"},{\"id\":276390136,\"identity\":\"1c69af7f-83a6-4f1e-8a7d-e0fd4a82140d\",\"order_by\":1,\"name\":\"Lisha Ying\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhejiang Cancer Institute, Zhejiang Cancer Hospital, Chinese Academy of Sciences\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Lisha\",\"middleName\":\"\",\"lastName\":\"Ying\",\"suffix\":\"\"},{\"id\":276390137,\"identity\":\"72100a79-ae99-47d6-9257-ba8bd995edf2\",\"order_by\":2,\"name\":\"Zhengxiao Ma\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital)\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhengxiao\",\"middleName\":\"\",\"lastName\":\"Ma\",\"suffix\":\"\"},{\"id\":276390138,\"identity\":\"b77415d8-9a75-4e51-83f6-12896b00d78e\",\"order_by\":3,\"name\":\"Ying Yang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhejiang Chinese Medicine University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ying\",\"middleName\":\"\",\"lastName\":\"Yang\",\"suffix\":\"\"},{\"id\":276390139,\"identity\":\"21f6504c-5883-4ca6-b6cd-c0523846d145\",\"order_by\":4,\"name\":\"Rui Zhu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Zhejiang Cancer Hospital, Chinese Academy of Sciences\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Rui\",\"middleName\":\"\",\"lastName\":\"Zhu\",\"suffix\":\"\"},{\"id\":276390140,\"identity\":\"63a27893-3b98-4a6b-bc3e-0b5254879413\",\"order_by\":5,\"name\":\"Dan Su\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYHACAwaGCgZmUrWcIVkLYxsp6uVnJG/8XDivjp2//QDj44pfDPLmBK24kVYsPXMbG7PEmQRmw7N9DIY7GwhpkcgxkObdxsNsIMHAJtnYw5BgcICgw3KMf/POkSBBC8ONHDNp3gYDiJaGH0RoMTjzrMya51gC0C+JzYaNDRKGGwg6rD15822emrpk/vbDBx82/LGRJ+wwKEhmYGBsAEaQBJHqgcAOQv0hXscoGAWjYBSMHAAAB4I3MwWbOi8AAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital)\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Dan\",\"middleName\":\"\",\"lastName\":\"Su\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-03-04 07:46:07\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4010987/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4010987/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":52195202,\"identity\":\"2659043f-708e-4012-9b29-622c6bb5c109\",\"added_by\":\"auto\",\"created_at\":\"2024-03-07 19:52:15\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":808610,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDifferential expression of genes between tumor and normal samples.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4010987/v1/86bf06a4aa01bc786c259d63.png\"},{\"id\":52195217,\"identity\":\"cb93130c-767b-4f99-9d1e-2f22e1d403c2\",\"added_by\":\"auto\",\"created_at\":\"2024-03-07 19:52:19\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":151657,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe distribution of EcDNAm in the tumor and matched normal samples. 1M, megabase; ge, greater than or equal; lt, less than.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4010987/v1/5e61956c24473fe6abe89b87.png\"},{\"id\":52195173,\"identity\":\"42b2e14e-66b5-4a19-9238-fcaca727a5ff\",\"added_by\":\"auto\",\"created_at\":\"2024-03-07 19:52:13\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":216972,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe distribution of the number of the gene count which derived from EcDNA molecules between LC and matched normal samples.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4010987/v1/83b8f1d756ba4f090a16083f.png\"},{\"id\":52195206,\"identity\":\"f2211730-320f-4863-9ec0-1552fa2ce085\",\"added_by\":\"auto\",\"created_at\":\"2024-03-07 19:52:17\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":70090,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe distribution of the driver oncogenes which derived from EcDNA molecules between LC and matched normal samples.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4010987/v1/93bbf8e4fb4746ee06489662.png\"},{\"id\":52195207,\"identity\":\"4ce2f150-d724-4429-9ab8-7462282c6444\",\"added_by\":\"auto\",\"created_at\":\"2024-03-07 19:52:17\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":481127,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGO analysis of genes in EcDNA molecules. GO, Gene Ontology.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4010987/v1/f8fc30d48665bede51895848.png\"},{\"id\":52195174,\"identity\":\"e205d00a-f806-47da-a69d-ba16ad82c5c1\",\"added_by\":\"auto\",\"created_at\":\"2024-03-07 19:52:13\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":594493,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eKEGG pathway analyses of genes in EcDNA molecules. KEGG, Kyoto Encyclopedia of Genes and Genomes.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4010987/v1/bde859bc08deb80724f888c5.png\"},{\"id\":52471615,\"identity\":\"b5730baa-5eca-4bdd-a391-35d93af286f5\",\"added_by\":\"auto\",\"created_at\":\"2024-03-12 03:36:46\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2807573,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4010987/v1/0b18643f-c221-4dfd-8999-1584ba68060d.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"The distribution of the extrachromosomal DNA molecules in early lung cancer\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eLung cancer (LC) has the highest morbidity and mortality rate in the world (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). Non-small cell lung cancer (NSCLC) accounts for three quarters of LC (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e). With the popularization of CT examination and low-dose spiral CT screening for LC, the number of patients with early NSCLC is increasing year by year (\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e). Compared to targeted therapy and immunotherapy for patients with advanced lung cancer, surgical therapy is the main treatment for patients with early LC, but the prognosis is different (\\u003cspan additionalcitationids=\\\"CR6\\\" citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e). According to the eighth edition of the staging classification, the 2-year recurrence and metastasis rate of surgically resected stage I NSCLC patients are 38%, and the 5-year overall survival rate is 60\\u0026ndash;74% (\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e). To improve the survival rate of patients with early NSCLC, it is particularly important to find potential biomolecular targets and to elucidate their roles and mechanisms in the process of recurrence and metastasis.\\u003c/p\\u003e \\u003cp\\u003eApart from 22 linear autosome pairs and a pair of sex chromosomes, extrachromosomal DNA molecules (EcDNAm) were found in the human genome (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e). With the explosive growth of next generation sequencing (NGS) biological data in recent years, EcDNAm has become a research hotspot in the field of biomedical oncology (\\u003cspan additionalcitationids=\\\"CR13 CR14\\\" citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e). EcDNAm were found to be a closed loop structure (16, 17). Wu e\\u003cem\\u003et al.\\u003c/em\\u003e reported that EcDNAm with longer sizes are often enriched in the process of tumor formation and aging, and participate in the occurrence and development of tumor aging in a special way (\\u003cspan additionalcitationids=\\\"CR19\\\" citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e). At the same time, the chromatin of EcDNAm with longer sizes is in a highly open state, in which the transcription of genes is abnormally active, and the mutation of the oncogenic driver gene mediates drug resistance (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). These findings indicated that EcDNAm plays an important role in tumorigenesis. However, the mechanism of action of EcDNAm in patients with early stage LC remains unclear.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we explored the mechanism of action of EcDNAm in early stage of LC. To the best of our knowledge, this is the first report to investigate the distribution, function, and mechanism of EcDNAm in matched early LC samples.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eTissue DNA preparation and extrachromosomal DNA molecules sequencing\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe study protocol was approved by the institutional review board at Zhejiang Cancer Hospital (IRB-2020-63). Written informed consent was obtained from two patients with early lung cancer at the Department of Thoracic Surgery at Zhejiang Cancer Hospital. Two tumor tissue samples and matched non-tumor lung cancer samples were collected during the surgery. High-throughput extrachromosomal DNA sequencing was performed by Novogene Biotech Inc. (Beijing, China). Extrachromosomal DNA purification and sequencing were performed according to a previously publication method(\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003ch3\\u003eSequencing analysis of extrachromosomal DNA molecules\\u003c/h3\\u003e\\n\\u003cp\\u003eSequencing data were obtained using an Illumina NovaSeq 600 sequencer in 150bp paired-end mode. After that, the raw sequencing data were further quality controlled; for instance, low-quality reads were discarded, and the adaptor was removed. Some bioinformatics tools, such as circle-map, samtools and bedtools, have been used to detect extrachromosomal DNA molecules. Gene function and pathway analysis were performed using the GSEA package.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eAll statistical analyses and diagram drawings were processed by the R software (version 4.1.2, R Foundation for Statistical Computing, Vienna, Austria). DEseq2 software was used to analyze the expression of 59 early lung cancer patients and their corresponding normal samples using RNA transcriptome data. \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered for the statistical significance.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eDifferential Expression between tumor and corresponding normal samples using RNA sequencing data\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo investigate the expression level of genes on linear chromosomes in patients with early lung cancer, we used transcriptome data (available from the published article: PMID\\u0026thinsp;=\\u0026thinsp;37694148) to analyze the expression of 59 early lung cancer patients and corresponding normal samples. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, we found a significant difference between the tumor and corresponding normal samples for some genes, such as EGFR. At the same time, obvious distinctions were observed between relapsed and non-relapsed tumor samples.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eGenome-wide detection of EcDNAm in paired LC samples\\u003c/h3\\u003e\\n\\u003cp\\u003eEcDNAm were detected in 23 pairs of chromosomes by mapping the clean reads to the genome. As showed in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, these results indicate that the presence of EcDNAm is common in tumors and matched normal samples.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe genomic distribution of EcDNAm revealed that it was common in all 23 pairs of chromosomes. Few EcDNAm were detected in the mitochondria.\\u003c/p\\u003e \\u003cp\\u003eAccording to the previous classification criteria (\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e), EcDNAm is generally divided into two types. One is extrachromosomal circular DNA (\\u003cb\\u003eEccDNA)\\u003c/b\\u003e, which is usually less than 1KB and cannot be seen under an optical microscope. EccDNA mainly includes telomere rings, small polydisperse DNA elements and micro-DNA. The other is extrachromosomal DNA (\\u003cb\\u003eEcDNA)\\u003c/b\\u003e, which is usually larger than 1 MB. EcDNA carries one or more oncogenic driver genes, and EcDNA is visible by light microscopy (\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). As shown In Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, the majority of the EcDNA was mainly focused on the tumor samples.\\u003c/p\\u003e\\n\\u003ch3\\u003eComparing the discrepancy between LC and matched normal samples in EcDNA\\u003c/h3\\u003e\\n\\u003cp\\u003eMost of the large EcDNA molecules (\\u0026gt;1 MB) were detected between the LC and matched normal samples. As showed in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, the number of genes contained in EcDNA molecules varied in chromosome between LC and matched normal samples. Previous studies have demonstrated that large EcDNA molecules carried one or multiple driver oncogenes (\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e). Thus, the distribution of driver oncogenes derived from EcDNA molecules was examined. Compared to the matched normal samples, the number of EcDNA molecules that contained driver oncogenes was relatively high (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eGO and KEGG pathway analysis of genes in EcDNA molecules\\u003c/h3\\u003e\\n\\u003cp\\u003eGO analysis was performed to examine the functions of genes associated with EcDNA molecules, such as related cellular components (CC), molecular functions (MF), and biological processes (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The dominant biological processes were related to the positive regulation of protein localization, axon development and in utero embryonic development in the tumor samples. The detailed results of gene function are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eGo analysis of the genes associated with EcDNA in paired early LC samples\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSample\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCategory\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eDescription\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eCase 1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003epositive regulation of protein localization\\u003c/p\\u003e \\u003cp\\u003eresponse to xenobiotic stimulus\\u003c/p\\u003e \\u003cp\\u003esmall GTPase mediated signal transduction\\u003c/p\\u003e \\u003cp\\u003eaxonogenesis\\u003c/p\\u003e \\u003cp\\u003eaxon development\\u003c/p\\u003e \\u003cp\\u003emononuclear cell differentiation\\u003c/p\\u003e \\u003cp\\u003eembryonic organ development\\u003c/p\\u003e \\u003cp\\u003eregulation of neuron projection development\\u003c/p\\u003e \\u003cp\\u003ein utero embryonic development\\u003c/p\\u003e \\u003cp\\u003esmall molecule catabolic process\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eglutamatergic synapse\\u003c/p\\u003e \\u003cp\\u003eneuronal cell body\\u003c/p\\u003e \\u003cp\\u003emitochondrial matrix\\u003c/p\\u003e \\u003cp\\u003ecell-substrate junction\\u003c/p\\u003e \\u003cp\\u003efocal adhesion\\u003c/p\\u003e \\u003cp\\u003ecollagen-containing extracellular matrix\\u003c/p\\u003e \\u003cp\\u003esynaptic membrane\\u003c/p\\u003e \\u003cp\\u003eneuron to neuron synapse\\u003c/p\\u003e \\u003cp\\u003ecell leading edge\\u003c/p\\u003e \\u003cp\\u003epostsynaptic specialization\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eMF\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eactin binding\\u003c/p\\u003e \\u003cp\\u003eprotein serine/threonine kinase activity\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eNormal 1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003esmall molecule catabolic process\\u003c/p\\u003e \\u003cp\\u003eregulation of anatomical structure size\\u003c/p\\u003e \\u003cp\\u003eregulation of cellular component size\\u003c/p\\u003e \\u003cp\\u003esmall GTPase mediated signal transduction\\u003c/p\\u003e \\u003cp\\u003eaxonogenesis\\u003c/p\\u003e \\u003cp\\u003eembryonic organ development\\u003c/p\\u003e \\u003cp\\u003eresponse to nutrient levels\\u003c/p\\u003e \\u003cp\\u003emitotic cell cycle phase transition\\u003c/p\\u003e \\u003cp\\u003eregulation of immune effector process\\u003c/p\\u003e \\u003cp\\u003eregulation of trans-synaptic signaling\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eglutamatergic synapse\\u003c/p\\u003e \\u003cp\\u003emitochondrial matrix\\u003c/p\\u003e \\u003cp\\u003eneuronal cell body\\u003c/p\\u003e \\u003cp\\u003epostsynaptic specialization\\u003c/p\\u003e \\u003cp\\u003easymmetric synapse\\u003c/p\\u003e \\u003cp\\u003ecell leading edge\\u003c/p\\u003e \\u003cp\\u003epostsynaptic density\\u003c/p\\u003e \\u003cp\\u003ecoated vesicle\\u003c/p\\u003e \\u003cp\\u003eneuron to neuron synapse\\u003c/p\\u003e \\u003cp\\u003enuclear speck\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eCase 2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eresponse to xenobiotic stimulus\\u003c/p\\u003e \\u003cp\\u003epositive regulation of protein localization\\u003c/p\\u003e \\u003cp\\u003eembryonic organ development\\u003c/p\\u003e \\u003cp\\u003ein utero embryonic development\\u003c/p\\u003e \\u003cp\\u003eaxon development\\u003c/p\\u003e \\u003cp\\u003eaxonogenesis\\u003c/p\\u003e \\u003cp\\u003eregulation of anatomical structure size\\u003c/p\\u003e \\u003cp\\u003edouble-strand break repair\\u003c/p\\u003e \\u003cp\\u003esmall GTPase mediated signal transduction\\u003c/p\\u003e \\u003cp\\u003eresponse to nutrient levels\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eglutamatergic synapse\\u003c/p\\u003e \\u003cp\\u003ecell-substrate junction\\u003c/p\\u003e \\u003cp\\u003efocal adhesion\\u003c/p\\u003e \\u003cp\\u003esynaptic membrane\\u003c/p\\u003e \\u003cp\\u003ecell leading edge\\u003c/p\\u003e \\u003cp\\u003eneuronal cell body\\u003c/p\\u003e \\u003cp\\u003ecollagen-containing extracellular matrix\\u003c/p\\u003e \\u003cp\\u003eneuron to neuron synapse\\u003c/p\\u003e \\u003cp\\u003epostsynaptic membrane\\u003c/p\\u003e \\u003cp\\u003evesicle lumen\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eMF\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eactin binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eNormal 2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003emononuclear cell differentiation\\u003c/p\\u003e \\u003cp\\u003elymphocyte differentiation\\u003c/p\\u003e \\u003cp\\u003eresponse to xenobiotic stimulus\\u003c/p\\u003e \\u003cp\\u003eaxonogenesis\\u003c/p\\u003e \\u003cp\\u003eembryonic organ development\\u003c/p\\u003e \\u003cp\\u003eresponse to nutrient levels\\u003c/p\\u003e \\u003cp\\u003ecell fate commitment\\u003c/p\\u003e \\u003cp\\u003eepidermis development\\u003c/p\\u003e \\u003cp\\u003ecytokine-mediated signaling pathway\\u003c/p\\u003e \\u003cp\\u003esmall GTPase mediated signal transduction\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eglutamatergic synapse\\u003c/p\\u003e \\u003cp\\u003ecell-substrate junction\\u003c/p\\u003e \\u003cp\\u003eneuron to neuron synapse\\u003c/p\\u003e \\u003cp\\u003efocal adhesion\\u003c/p\\u003e \\u003cp\\u003epostsynaptic specialization\\u003c/p\\u003e \\u003cp\\u003emitochondrial matrix\\u003c/p\\u003e \\u003cp\\u003easymmetric synapse\\u003c/p\\u003e \\u003cp\\u003esynaptic membrane\\u003c/p\\u003e \\u003cp\\u003epostsynaptic density\\u003c/p\\u003e \\u003cp\\u003ecollagen-containing extracellular matrix\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eMF\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eprotein serine/threonine kinase activity\\u003c/p\\u003e \\u003cp\\u003eactin binding\\u003c/p\\u003e \\u003cp\\u003eDNA-binding transcription factor binding\\u003c/p\\u003e \\u003cp\\u003eprotein serine kinase activity\\u003c/p\\u003e \\u003cp\\u003eRNA polymerase II-specific DNA-binding transcription factor binding\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003ctfoot\\u003e \\u003ctr\\u003e\\u003ctd colspan=\\\"3\\\"\\u003eBP, Biological Process; CC, Cellular Component; MF, Molecular Function\\u003c/td\\u003e\\u003c/tr\\u003e \\u003c/tfoot\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn addition, KEGG pathway analysis indicated that the main pathway was involved in the cell cycle which departed from normal samples (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eAn extrachromosomal DNA molecule (EcDNAm) is circular DNA that plays an important role in the development and heterogeneity of cancer (\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e). However, the structure, composition and genome-wide frequency of extrachromosomal DNA have not been extensively profiled. In this study, high-throughput sequencing demonstrated the presence of EcDNAm in paired early LC samples, in agreement with previous studies of other cancer types (\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e). In contrast, our data demonstrated that EcDNAm could be found in human genome, with the exception of mitochondria, in early lung cancer.\\u003c/p\\u003e \\u003cp\\u003eBased on size, two classes of EcDNAm exist in human cells: small (EccDNA) and large (EcDNA). Previous studies have shown that oncogenes in EcDNA are highly expressed (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e). Compared with the matched normal samples, the number of EcDNAm with a longer size (namely EcDNA), which contained driver oncogenes (for instance, EGFR) was relatively high. Furthermore, the majority of EcDNA was mainly focused on tumor sample, and no obvious differences were found between LC and matched normal samples in this study.\\u003c/p\\u003e \\u003cp\\u003eGO analysis of genes associated with EcDNA showed that the dominant biological processes were related to positive regulation of protein localization, axon development and in utero embryonic development in tumor samples. KEGG pathway analysis of genes associated with EcDNA indicated that the main pathway was involved in the cell cycle, which departed from normal samples. In this study, we found a significant difference between tumor and corresponding normal samples for some genes, such as EGFR. Based on the limited data and experimental validation, the carcinogenic mechanism of the relationship between chromosomal DNA and extra-chromosomal DNA remains unclear.\\u003c/p\\u003e \\u003cp\\u003eIn the future, we will increase the sample size and further explore the functional mechanism of EcDNAm in early LC. We believe that our findings fill the investigation of the EcDNAm gap in early LC, and that future studies should focus on the application of EcDNA as a potential biomarker and therapeutic target in patients with early LC.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eIn conclusion, our study confirmed the genome-wide presence of EcDNAm in parired LC samples. In addition, our work revealed the potential mechanisms of EcDNAm in early LC. This work provides further insights into our understanding of genome plasticity and the role of EcDNAm in early LC, and may contribute to the development of potential clinical therapies.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor\\u0026rsquo;s contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eJianfei Fang and Dan Su conceived, designed, and revised the study. Jianfei Fang analyzed the data and wrote the manuscript. Lisha Ying, Zhengxiao Ma, Ying Yang and Rui Zhu analyzed the data. All authors have read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study protocol was approved by the institutional review board at Zhejiang Cancer Hospital (IRB-2020-63). Written informed consent was obtained from two patients with early lung cancer at the Department of Thoracic Surgery at Zhejiang Cancer Hospital. This study was performed in line with the principles of the Declaration of Helsinki.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was supported by the Natural Science Foundation of Zhejiang Province, China (No.LQ21F010001).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was supported by the Natural Science Foundation of Zhejiang Province, China (No.LQ21F010001).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated during and/or analyzed during the current study are available in the GSA for Human (https://ngdc.cncb.ac.cn.gsa-huaman/), with access numbers PRJCA010105. The extrachromosomal sequencing datasets are not publicly available but are available from the corresponding author upon reasonable request.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot application\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor details\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003e1\\u003c/sup\\u003eDepartment of Pathology, Zhejiang Cancer Hospital, Hangzhou Institute of Medicine(HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003e2\\u003c/sup\\u003eZhejiang Cancer Institute, Zhejiang Cancer Hospital, Hangzhou Institute of Medicine(HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003e3\\u003c/sup\\u003ePostgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital) , Hangzhou, Zhejiang, 310022, China.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003e4\\u003c/sup\\u003eThe Second Clinical Medical College, Zhejiang Chinese Medicine University, Hangzhou, Zhejiang 310053, China\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eTeixeira VH, Pipinikas CP, Pennycuick A, Lee-Six H, Chandrasekharan D, Beane J, et al. Deciphering the genomic, epigenomic, and transcriptomic landscapes of pre-invasive lung cancer lesions. Nature medicine. 2019;25(3):517-25.\\u003c/li\\u003e\\n\\u003cli\\u003eChen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics in China, 2015. CA: a cancer journal for clinicians. 2016;66(2):115-32.\\u003c/li\\u003e\\n\\u003cli\\u003eTorre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA: a cancer journal for clinicians. 2015;65(2):87-108.\\u003c/li\\u003e\\n\\u003cli\\u003ePeng S, Li X, Wang Y, Liu J. [Effect of Adjuvant Chemotherapy on DFS for Patients with Stage I NSCLC]. Zhongguo fei ai za zhi = Chinese journal of lung cancer. 2017;20(7):485-9.\\u003c/li\\u003e\\n\\u003cli\\u003eRemon J, Besse B, Soria JC. Erratum to: Successes and failures: what did we learn from recent first-line treatment immunotherapy trials in non-small cell lung cancer? BMC medicine. 2017;15(1):82.\\u003c/li\\u003e\\n\\u003cli\\u003eCancer Genome Atlas Research N. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511(7511):543-50.\\u003c/li\\u003e\\n\\u003cli\\u003eCancer Genome Atlas Research N. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012;489(7417):519-25.\\u003c/li\\u003e\\n\\u003cli\\u003eSu S, Scott WJ, Allen MS, Darling GE, Decker PA, McKenna RJ, et al. Patterns of survival and recurrence after surgical treatment of early stage non-small cell lung carcinoma in the ACOSOG Z0030 (ALLIANCE) trial. The Journal of thoracic and cardiovascular surgery. 2014;147(2):747-52: Discussion 52-3.\\u003c/li\\u003e\\n\\u003cli\\u003eChansky K, Detterbeck FC, Nicholson AG, Rusch VW, Vallieres E, Groome P, et al. The IASLC Lung Cancer Staging Project: External Validation of the Revision of the TNM Stage Groupings in the Eighth Edition of the TNM Classification of Lung Cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2017;12(7):1109-21.\\u003c/li\\u003e\\n\\u003cli\\u003eCox D, Yuncken C, Spriggs AI. Minute Chromatin Bodies in Malignant Tumours of Childhood. Lancet. 1965;1(7402):55-8.\\u003c/li\\u003e\\n\\u003cli\\u003eZuo S, Yi Y, Wang C, Li X, Zhou M, Peng Q, et al. Extrachromosomal Circular DNA (eccDNA): From Chaos to Function. Frontiers in cell and developmental biology. 2021;9:792555.\\u003c/li\\u003e\\n\\u003cli\\u003eLuo J, Li Y, Zhang T, Xv T, Chen C, Li M, et al. Extrachromosomal circular DNA in cancer drug resistance and its potential clinical implications. Frontiers in oncology. 2022;12:1092705.\\u003c/li\\u003e\\n\\u003cli\\u003eYang L, Jia R, Ge T, Ge S, Zhuang A, Chai P, et al. Extrachromosomal circular DNA: biogenesis, structure, functions and diseases. Signal transduction and targeted therapy. 2022;7(1):342.\\u003c/li\\u003e\\n\\u003cli\\u003eZhao Y, Yu L, Zhang S, Su X, Zhou X. Extrachromosomal circular DNA: Current status and future prospects. eLife. 2022;11.\\u003c/li\\u003e\\n\\u003cli\\u003eChen Y, Qiu Q, She J, Yu J. Extrachromosomal circular DNA in colorectal cancer: biogenesis, function and potential as therapeutic target. Oncogene. 2023;42(13):941-51.\\u003c/li\\u003e\\n\\u003cli\\u003eSun Z, Ji N, Zhao R, Liang J, Jiang J, Tian H. Extrachromosomal circular DNAs are common and functional in esophageal squamous cell carcinoma. Annals of translational medicine. 2021;9(18):1464.\\u003c/li\\u003e\\n\\u003cli\\u003eLi R, Wang Y, Li J, Zhou X. Extrachromosomal circular DNA (eccDNA): an emerging star in cancer. Biomarker research. 2022;10(1):53.\\u003c/li\\u003e\\n\\u003cli\\u003eTurner KM, Deshpande V, Beyter D, Koga T, Rusert J, Lee C, et al. Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity. Nature. 2017;543(7643):122-5.\\u003c/li\\u003e\\n\\u003cli\\u003eWu S, Turner KM, Nguyen N, Raviram R, Erb M, Santini J, et al. Circular ecDNA promotes accessible chromatin and high oncogene expression. Nature. 2019;575(7784):699-703.\\u003c/li\\u003e\\n\\u003cli\\u003eBarreto SC, Uppalapati M, Ray A. Small Circular DNAs in Human Pathology. The Malaysian journal of medical sciences : MJMS. 2014;21(3):4-18.\\u003c/li\\u003e\\n\\u003cli\\u003eNathanson DA, Gini B, Mottahedeh J, Visnyei K, Koga T, Gomez G, et al. Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA. Science. 2014;343(6166):72-6.\\u003c/li\\u003e\\n\\u003cli\\u003eVerhaak RGW, Bafna V, Mischel PS. Extrachromosomal oncogene amplification in tumour pathogenesis and evolution. Nature reviews Cancer. 2019;19(5):283-8.\\u003c/li\\u003e\\n\\u003cli\\u003eMorton AR, Dogan-Artun N, Faber ZJ, MacLeod G, Bartels CF, Piazza MS, et al. Functional Enhancers Shape Extrachromosomal Oncogene Amplifications. Cell. 2019;179(6):1330-41 e13.\\u003c/li\\u003e\\n\\u003cli\\u003eDong Y, He Q, Chen X, Yang F, He L, Zheng Y. Extrachromosomal DNA (ecDNA) in cancer: mechanisms, functions, and clinical implications. Frontiers in oncology. 2023;13:1194405.\\u003c/li\\u003e\\n\\u003cli\\u003edeCarvalho AC, Kim H, Poisson LM, Winn ME, Mueller C, Cherba D, et al. Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma. Nature genetics. 2018;50(5):708-17.\\u003c/li\\u003e\\n\\u003cli\\u003eMoller HD, Mohiyuddin M, Prada-Luengo I, Sailani MR, Halling JF, Plomgaard P, et al. Circular DNA elements of chromosomal origin are common in healthy human somatic tissue. Nature communications. 2018;9(1):1069.\\u003c/li\\u003e\\n\\u003cli\\u003eKumar P, Dillon LW, Shibata Y, Jazaeri AA, Jones DR, Dutta A. Normal and Cancerous Tissues Release Extrachromosomal Circular DNA (eccDNA) into the Circulation. Molecular cancer research : MCR. 2017;15(9):1197-205.\\u003c/li\\u003e\\n\\u003cli\\u003eZhang P, Mbodj A, Soundiramourtty A, Llauro C, Ghesquiere A, Ingouff M, et al. Extrachromosomal circular DNA and structural variants highlight genome instability in Arabidopsis epigenetic mutants. Nature communications. 2023;14(1):5236.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"extrachromosomal DNA molecules (EcDNAm), lung cancer(LC), high throughput sequencing, EcDNA, bioinformatics\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4010987/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4010987/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eLung cancer (LC) is a cancer with high mortality worldwide. Research on the distribution and nature of extrachromosomal DNA molecules (EcDNAm) in early LC is scarce.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e \\u003cp\\u003eAfter removing linear DNA and mitochondrial circular DNA, EcDNAm were extracted from two paired LC tissue samples and amplified using rolling circle amplification. High throughput extrachromosomal DNA or RNA sequencing and bioinformatics analysis were used to explore the distribution and nature of the EcDNAm. To learn more about the role of oncogenes with large EcDNAm sizes, gene onology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eRNA sequencing results revealed the significant difference in some genes between tumor and corresponding normal samples. At the same time, obvious distinctions were observed between relapsed and non-relapsed tumor samples. The nature of the EcDNAm was comparable between LC samples and matched normal samples. Compared with the matched normal samples, the number of EcDNAm with longer size (EcDNA), which contained driver oncogenes, was relatively high. The majority of EcDNA in this study was mainly focused on the tumor samples. Enrichment analysis of the cancer samples revealed enrichment in biological processes, such as positive regulation of protein localization, axon development and in utero embryonic development.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e \\u003cp\\u003eThis study demonstrated the universality of the distribution and described the nature of EcDNAm in early LC. Moreover, our work fills the investigation of the EcDNAm gap and future studies should focus on the application of EcDNA as a potential biomarker in patients with early LC.\\u003c/p\\u003e\",\"manuscriptTitle\":\"The distribution of the extrachromosomal DNA molecules in early lung cancer\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-03-07 19:51:17\",\"doi\":\"10.21203/rs.3.rs-4010987/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"7f4522bd-5ec5-4ae6-ba9f-c6c9988fc1e7\",\"owner\":[],\"postedDate\":\"March 7th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-03-12T03:36:06+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-03-07 19:51:17\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4010987\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4010987\",\"identity\":\"rs-4010987\",\"version\":[\"v1\"]},\"buildId\":\"iFTdqyg4nuje_uAy1AHro\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}