Extrachromosomal DNAs (ecDNAs) are circular DNA structures located in the nuclei of cells outside chromosomes. They were originally discovered in chromosome spreads of cells obtained from embryonal tumors in the 1960s and were called “double minutes” because of their minuscule appearance.1 Since then, molecular biologic and genome technologies have shown ecDNAs to be a driver of carcinogenesis, tumor heterogeneity, genome instability (see Key Concepts), immune evasion, and therapy resistance.
Proposed mechanisms for the formation of ecDNAs include complex chromosomal-shattering and genome-rearrangement events. The ecDNAs lack centromeres and originate from chromosomal loci that can contain oncogenes, regulatory elements, and immunomodulatory genes. The current understanding is that ecDNAs are unique to cancer cells and are not found in healthy cells.2 The segregation of ecDNA into daughter cancer cells is random and uneven, which creates opportunities for genetic advantage to accrue to one daughter cell over the other (Figure 1). Recent studies revealed the landscape of ecDNA in human cancer3 and showed that coordinated inheritance of specialized ecDNA combinations can occur under selective pressures, such as cancer treatment.4 In addition, the investigators found that clusters of cooperative ecDNA (called hubs) in the nucleus facilitate the efficient transcription of genes in ecDNA and on chromosomes, which further promotes the aggressive behavior of tumor cells (Figure 1).3,4
FIGURE 1
Biologic Features of Extrachromosomal DNAs.
Bailey et al. analyzed the genomes of 15,382 tumor samples that had been obtained from 14,778 patients enrolled in the Genomics England 100,000 Genomes Project. On reconstruction of focally amplified regions in these genomes, they detected ecDNA in 17.1% of the tumor samples — most commonly from sarcoma, glioblastoma, gastroesophageal adenocarcinoma, human epidermal growth factor receptor 2 (HER2)–positive breast cancer, and squamous-cell carcinoma of the lung. These results were consistent with those from a previous analysis of the Cancer Genome Atlas, in which 14.3% of the tumors carried ecDNA.5
Bailey et al. found that the genes most commonly seen in ecDNA included those involved in controlling the cell cycle (CCND1 and CDK4), signal transduction (EGFR, ERBB2, FGFR1, and KRAS), and master regulation (MYC, MDM2, and MDM4). Oncogenes in ecDNAs were present in higher copy numbers than were seen in focal amplifications on conventional chromosomes. Tumor samples with ecDNAs carrying immunomodulatory genes, some of which suppress lymphocyte activation and immune effector processes, were found to have fewer T cells than tumor samples with ecDNAs containing oncogenes without immunomodulatory genes. Correlates of genome instability, including the duplication of entire genomes, a high burden of structural variants, and damaging variants in specific tumor-suppressor genes such as TP53, were associated with the presence of ecDNA, as was exposure to cytotoxic and targeted agents. Multilocus sampling of tumor tissues showed heterogeneous intratumoral distribution of ecDNA. In a finding consistent with dynamic selection and multiple roles in sculpting the cancer genome and immune microenvironment, the presence of ecDNAs was associated with a poor prognosis, as evidenced by a more-advanced tumor stage, the development of metastases, and shorter overall survival.
Microscopy-based karyotyping methods such as DNA fluorescence in situ hybridization are the standard clinical methods to visualize ecDNA, but they are low-throughput and rely on the use of targeted probes. In contrast, computational tools and algorithms, such as those used by Bailey et al., can be applied agnostically to large-scale genome-sequencing data sets to reconstruct focally amplified regions in order to infer ecDNA structures and content. Emerging technological innovations may complement the analysis of short-read sequences. New library enrichment methods can be deployed before sequencing to optimize ecDNA detection. Long-read sequencing, which produces sequence reads of tens to hundreds of thousands of DNA base pairs, offers the means to deconvolute structural heterogeneities. At present, clinical whole-genome sequencing, which is ideally complemented by RNA sequencing, represents the most direct path to the precise detection and interpretation of pathogenic elements on ecDNAs.
Studies such as the one conducted by Bailey at al. fuel interest in targeting extrachromosomal structures as a strategy to treat cancer. Although many ecDNA-encoded oncogenes and their protein products, such as epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1), are validated drug targets for therapeutic intervention, others remain challenging and are not yet amenable to modulation by anticancer agents. Furthermore, intratumoral heterogeneity and the development of adaptive resistance can minimize the therapeutic benefits of approaches involving the targeting of genes in ecDNA (or the proteins they encode). Thus, targeting the biogenesis and actions of ecDNAs by leveraging their unique characteristics is appealing.
For instance, one strategy is to inhibit specific proteins that tether ecDNA within ecDNA hubs.2 Perhaps the most exciting strategy to interfere with ecDNA-mediated carcinogenesis involves an inhibitor of checkpoint kinase 1 (CHK1) that is being investigated in a phase 1 study for use as monotherapy or in combination with targeted agents in tumors with oncogene amplifications (ClinicalTrials.gov number, NCT05827614). Tumor cells that contain ecDNA are under replication stress, which leads to DNA damage of ecDNAs and an increase in CHK1 activity, thus promoting cell-cycle arrest. CHK1 inhibitors release the brakes on cell-cycle arrest, rendering the DNA-damaged ecDNA-containing cancer cells vulnerable to apoptosis.6 Efforts to monitor ecDNA levels noninvasively in blood, cerebrospinal fluid, or other biologic fluids could offer a means of monitoring responses to these strategies. Thus, ecDNA, although known to oncologists for more than half a century, is gaining momentum as a target for intervention in the treatment of cancer.
Key Concepts
Genome instabilityA hallmark of cancer development and progression characterized by the accumulation of DNA damage; genome instability manifests as increased genetic variation across the genome, including chromosomal translocations, structural variants, extrachromosomal DNAs, and aneuploidy. Replication stress, frequently observed in rapidly dividing cells, and exogenous factors, such as ultraviolet and ionizing radiation, tobacco smoke, and certain chemicals, can result in genome instability. Early genetic events in the life of cancer cells, such as changes in the maintenance of telomeric length, or the activation of an oncogene can lead to genome instability. Loss-of-function variants in genes encoding tumor suppressor proteins, such as those that mediate DNA repair and cell-cycle checkpoints, can also contribute to an unstable genome.
OncogeneA gene that encodes an RNA or protein that can directly or indirectly cause cancer, often due to gain-of-function variants within it, amplification, or changes in its regulation, which can occur on chromosomal translocation. Oncogene activation may lead to uncontrolled cell growth and division, perturbed metabolism, enhanced metastasis, and evasion of cell death, all of which are considered hallmarks of cancer. Some specific variants in oncogenes function as drivers because they represent key and dominant genetic alterations that drive malignant processes. Anticancer agents have been developed to inhibit the actions of many oncogenes.
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