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Sci Prog
Sci Prog
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Science Progress
0036-8504
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SAGE Publications Sage UK: London, England

39228317
10.1177/00368504241276771
10.1177_00368504241276771
Medicine & Health Sciences
The distribution of the extrachromosomal DNA molecules in early lung cancer
Fang Jianfei 1
Ying Lisha 2
https://orcid.org/0009-0001-2379-489X
Ma Zhengxiao 3
Yang Ying 4
Zhu Rui 1
Su Dan 13
1 Department of Pathology, 89680 Zhejiang Cancer Hospital , Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang, China
2 Zhejiang Cancer Institute, 89680 Zhejiang Cancer Hospital , Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang, China
3 Postgraduate Training Base Alliance of Wenzhou Medical University (89680 Zhejiang Cancer Hospital ), Hangzhou, Zhejiang, China
4 The Second Clinical Medical College, Zhejiang Chinese Medicine University, Hangzhou, Zhejiang, China
Dan Su, Department of Pathology, Zhejiang Cancer Hospital, Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China. Email: sudan@zjcc.org.cn
4 9 2024
Jul-Sep 2024
107 3 00368504241276771© The Author(s) 2024
2024
SAGE Publications
https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage).
Lung cancer (LC) is a highly lethal cancer worldwide. Research on the distribution and nature of extrachromosomal DNA molecules (EcDNAm) in early LC is scarce. In this study, 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 (EcDNA) or RNA sequencing and bioinformatics analysis were subsequently utilized to explore the distribution and nature of the EcDNAm. Additionally, to elucidate the role of oncogenes with large EcDNAm sizes, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed. The RNA sequencing results revealed significant differences in certain genes between tumors and corresponding normal samples. At the same time, slight distinctions were observed between relapsed and non-relapsed tumor samples. The nature of the EcDNAm was compared between LC samples and matched normal samples. There was a tendency for the number of EcDNAm with longer size (EcDNA) and its containing driver oncogenes to be higher in cancer 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. This study highlights the universal distribution and characteristics 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
Zhejiang Provincial Natural Science Foundation LQ21F010001 typesetterts19
cover-dateJuly-September 2024
==== Body
pmcIntroduction

Lung cancer (LC) has the highest morbidity and mortality rate in the world.1,2 Non-small cell LC (NSCLC) accounts for three-quarters of all LC cases. 3 With the popularization of computed tomography (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 LC, 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 is 38%, and the 5-year overall survival rate is 60% to 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 biological data in recent years, EcDNAm have become a research hotspot in the field of biomedical oncology.12–15 EcDNAm were found to be a closed loop structure.16,17 Wu et 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 play 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 the 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 EcDNAm sequencing

The study protocol was approved by the institutional review board at Zhejiang Cancer Hospital, Hangzhou, China (IRB-2020-63). Written informed consent was obtained from two male patients with early stage LC undergoing surgery at the Department of Thoracic Surgery, Zhejiang Cancer Hospital. The samples were collected in March 2022 at Zhejiang Cancer Hospital, Hangzhou, China. High throughput extrachromosomal DNA (EcDNA) sequencing was performed by Novogene Biotech Inc. (Beijing, China). The method of circular DNA purification is the Circle-Seq protocol, which involves column purification, removal of remaining linear chromosomal DNA, rolling circle amplification, sequencing, and mapping.22,23 The detailed EcDNA purification and sequencing were performed according to a previously published method.16,22

Sequencing analysis of EcDNAm

Sequencing data were obtained using an Illumina NovaSeq 6000 sequencer in 150 bp paired-end mode. The fragmented DNA was subjected to library preparation with NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA). Subsequently, the raw sequencing data were further quality-controlled by Cutadapt software (v1.9.1). For instance, low-quality reads (Q < 10; reads containing more than 10% of the total length of N base) were discarded, and the adaptor sequences were removed. The clean reads were further aligned to the reference genome (UCSC hg19). Various bioinformatics tools, such as circle-map,24,25 samtools, 26 and bedtools, 26 have been used to detect EcDNAm. Gene function and pathway analyses were performed in a single sample using the clusterProfiler package (version 4.2.0).

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 (version 1.26.0) was used to analyze the expression of 59 early LC 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

RNA sequencing data and EcDNAm sequencing data were performed to explore the expression levels of an oncogene on linear chromosomes and extra-chromosomes in patients with early LC. In this part, to investigate the gene expression patterns on linear chromosomes in patients with early LC, we used transcriptome data 27 to assess the expression profiles of 59 early LC patients and their corresponding normal samples. As shown in Figure 1, we found a difference between the tumor and corresponding normal samples for certain genes, such as epidermal growth factor receptor (EGFR). At the same time, slight distinctions were observed between relapsed and non-relapsed tumor samples.

Figure 1. Differential expression of genes between tumor and normal 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 illustrated in Figure 2, these results indicate that the presence of EcDNAm is prevalent in both tumor and matched normal samples.

Figure 2. The distribution of EcDNAm in the tumor and matched normal samples.

EcDNAm: extrachromosomal DNA molecules; 1M: megabase; ge: greater than or equal; lt: less than; C1: sample 1 tumor sample; N1: sample 1 normal sample; C2: sample 2 tumor sample; N2: sample 2 normal sample.

The genomic distribution analysis of EcDNAm revealed its widespread presence across all 23 pairs of chromosomes. Few EcDNAm were detected in the mitochondria.

According to the previous classification criteria, 28 EcDNAm is generally divided into two types. One type is extrachromosomal circular DNA (EccDNA), typically <1 kB and invisible under an optical microscope. EccDNA primarily encompasses telomere rings, small polydisperse DNA elements, and micro-DNA. The other type is EcDNA, typically >1 MB, carrying one or more oncogenic driver genes, and EcDNA is visible by light microscopy. 28 Compared to the matched normal samples, there was a tendency for the quantity of EcDNA molecules to be higher in cancer samples (Figure 2).

Comparing the discrepancy in EcDNA between LC and matched normal samples

The majority of the large EcDNAm (>1 MB) were detected in both the LC and matched normal samples. As depicted in Figure 3, the number of genes within EcDNAm varied in chromosomes between LC and matched normal samples. Previous studies have demonstrated that large EcDNAm harbor one or multiple driver oncogenes.18,28,29 Consequently, the distribution of driver oncogenes derived from EcDNAm was examined. Compared to the matched normal samples, there was a trend for the number of EcDNAm that contained driver oncogenes to be higher in cancer samples (Figure 4).

Figure 3. The distribution of the number of gene counts derived from EcDNAm between LC and matched normal samples.

EcDNAm: extrachromosomal DNA molecules; LC: lung cancer.

Figure 4. The distribution of the driver oncogenes derived from EcDNAm between LC and matched normal samples.

EcDNAm: extrachromosomal DNA molecules; LC: lung cancer.

Gene ontology and Kyoto encyclopedia of genes and genomes pathway analysis of genes in EcDNAm

Gene ontology (GO) analysis was performed to examine the function of genes associated with EcDNAm, focusing on related cellular components (CCs), molecular functions (MFs), and biological processes (BPs), as outlined in Table 1. The predominant biological processes were related to the positive regulation of protein localization, axon development, and in-utero embryonic development in the tumor samples. Detailed results of gene functions are shown in Figure 5.

Figure 5. GO analysis of genes in EcDNAm.

GO: gene ontology: EcDNAm: extrachromosomal DNA molecules.

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 the 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	
GO: gene ontology: EcDNAm: extrachromosomal DNA molecules; BP: biological process; CC: cellular component; MF: molecular function.

In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the primary pathway was involved in the cell cycle indicating a deviation from the patterns observed in normal samples (Figure 6).

Figure 6. KEGG pathway analyses of genes in EcDNAm.

KEGG: Kyoto encyclopedia of genes and genomes; EcDNAm: extrachromosomal DNA molecules.

Discussion

An EcDNAm is a circular DNA that plays an important role in the development and heterogeneity of cancer.13,30,31 However, the comprehensive profiling of the structure, composition, and genome-wide frequency of EcDNA has not been extensively profiled. In this study, high throughput sequencing demonstrated the presence of EcDNAm in paired early LC samples, consistent with previous studies in other cancer types.32,33 In addition, our data demonstrated the presence of EcDNAm could be found in the human genome, except mitochondria, in early LC.

To investigate the expression levels of an oncogene on linear chromosomes and extra-chromosomes in patients with early LC, RNA sequencing data and EcDNAm sequencing data were performed in this study. The RNA sequencing results revealed differential genes between tumor and normal samples, such as EGFR.

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.19,34 In comparison with the corresponding normal samples, there was a trend for the number of EcDNAm with a larger size (namely EcDNA), which contains driver oncogenes (e.g. EGFR) was relatively elevated. Furthermore, there was a tendency for the quantity of EcDNA to be higher in tumor samples, although no statistical analysis has been conducted to confirm the difference.

GO analysis of genes associated with EcDNA showed that the predominant biological processes were related to positive regulation of protein localization, axon development, and in-utero embryonic development in tumor samples. Additionally, KEGG pathway analysis of genes associated with EcDNA indicated a primary involvement in the cell cycle, diverging from normal samples. In this study, we found a difference between tumor and corresponding normal samples for certain genes, such as EGFR. However, due to the limited data and experimental validation, the carcinogenic mechanism of the relationship between chromosomal DNA and EcDNA remains unclear. Furthermore, a direct statistical comparison of oncogenes associated with EcDNA was not performed between the LC samples and matched normal samples. We firmly believe that the oncogenes (such as EGFR) associated with EcDNA might play a crucial role in early LC.

In the future, we will expand the sample size and delve deeper into the functional mechanism of EcDNAm in early LC. We believe that our findings address the current gap in the understanding of EcDNAm in early LC, and we advocate for future studies to emphasize the potential application of EcDNA as a biomarker and therapeutic target in patients with early LC.

Conclusions

In conclusion, our study confirmed the genome-wide presence of EcDNAm in paired 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, contributing to the development of potential clinical therapies.

Author contributions: JF and DS conceived, designed, and revised the study. JF analyzed the data and wrote the manuscript. LY, ZM, YY, and RZ analyzed the data. All authors have read and approved the final manuscript.

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.

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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 LC at the Department of Thoracic Surgery at Zhejiang Cancer Hospital. This study was performed in line with the principles of the Declaration of Helsinki.

Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Zhejiang Provincial Natural Science Foundation, (grant number LQ21F010001).

ORCID iD: Zhengxiao Ma https://orcid.org/0009-0001-2379-489X
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