A Novel Class of DNA. Extrachromosomal circular DNA (eccDNA) consists of circular DNA fragments that exist outside the chromosomes. While most DNA is organized linearly on chromosomes, eccDNA forms closed circular molecules that are produced through DNA damage, chromothripsis, and structural genomic variations. Its presence in tumor tissue has been known since 1965.
Cancer Biology Relevance. In cancer cells, large eccDNA (called double minutes) plays a crucial role in oncogene amplification, enabling cancer cells to rapidly boost expression of growth-promoting genes without the constraints of chromosomal inheritance. Because eccDNA lacks centromeres, it is segregated randomly during cell division, generating tumor heterogeneity and facilitating cancer evolution.
eccDNA and EGFR Resistance. Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) with an 80% objective response rate in EGFR-mutated NSCLC. Despite strong initial responses, resistance develops in nearly all patients. Studies have proposed that amplification of EGFR on eccDNA is one mechanism through which cancer cells evade EGFR-TKI treatment.
Gap in Knowledge. Most eccDNA research has been conducted in cancer cell lines or tumor tissue, not in plasma. This study represents one of the first investigations of plasma eccDNA in a clinically homogenous cohort of NSCLC patients, examining how circulating eccDNA changes during osimertinib treatment and whether it predicts survival outcomes.
Patient Cohort. The study enrolled 32 patients with EGFR-mutated advanced NSCLC from four Danish hospitals, along with five healthy individuals as controls. Plasma samples were collected before osimertinib treatment (baseline) and during treatment at a median of 28 days after initiation. The majority of patients (81.3%) received osimertinib as second-line treatment after progression on erlotinib.
eccDNA Enrichment Protocol. Cell-free DNA (cfDNA) was isolated from a median of 3.8 mL of plasma. Mitochondrial DNA was removed using the MssI restriction enzyme, and linear DNA was degraded using an exonuclease, leaving only circular DNA intact. The remaining eccDNA was amplified using multiple displacement amplification before sequencing.
Next-Generation Sequencing. The enriched eccDNA library was sequenced on an Illumina NovaSeq 6000 at 30 million read pairs per sample using 151-bp paired-end sequencing. The Circle_Finder algorithm was then used to identify eccDNA molecules by detecting split reads that map to three genomic sites -- the characteristic signature of a circular DNA junction.
EGFR-Overlapping eccDNA Definition. eccDNA molecules containing any portion of the EGFR gene (located on chromosome 7, positions 55,019,017 to 55,211,628) were classified as EGFR-overlapping eccDNA. These were normalized to the total eccDNA count per patient to enable fair comparisons across samples with different overall eccDNA levels.
eccDNA Detected in All Samples. The Circle_Finder algorithm identified a median of 44,475 eccDNA molecules per patient at baseline (range 6,463 to 276,742). eccDNA was detected in all 32 cancer patients and all 5 healthy controls, confirming that circulating eccDNA is not exclusive to cancer. However, cancer patients had significantly more eccDNA than healthy controls at both baseline (p less than 0.001) and during treatment (p = 0.0012).
Size Distribution: Nucleosomal Peaks. The eccDNA showed a distinctive bimodal size distribution, with peaks corresponding to the size of one or two nucleosomes (approximately 147 and 294 bp plus DNA linker sequences). The vast majority (97.3% to 99.8%) of plasma eccDNA was below 2000 bp. This nucleosomal-sized pattern suggests that eccDNA remains wrapped around histone proteins in plasma, forming stable circular structures.
GC Content and Genomic Distribution. eccDNA had a higher GC content (median 45.1%) compared to random genomic fragments (40.9%), indicating a preference for GC-rich regions in eccDNA formation. The eccDNA was distributed across exonic, intronic, and intergenic regions without enrichment of any specific genomic elements, and was found on all chromosomes.
Treatment Response and Overall eccDNA Levels. After a median of 28 days on osimertinib, 73.3% of patients showed a decrease in total eccDNA levels. Patients with decreasing eccDNA had numerically longer progression-free survival (508 vs 154 days) and overall survival (955 vs 305 days) than those with increasing eccDNA, though these differences did not reach statistical significance, likely due to the small sample size.
Widespread Detection. EGFR-overlapping eccDNA was detected in 90.6% of baseline samples and 90.0% of response samples from cancer patients, and in 60% of healthy control samples. This high detection rate suggests that eccDNA containing EGFR sequences is a common feature of NSCLC plasma, not an artifact of the sequencing process.
Proportion Increases During Treatment. While the absolute number of EGFR-overlapping eccDNA did not significantly change from baseline to response samples, the proportion of EGFR-overlapping eccDNA relative to total eccDNA was significantly higher in response samples (p = 0.033). This suggests that under osimertinib treatment pressure, eccDNA containing EGFR sequences may be selectively maintained or enriched.
Key Finding: Survival Association During Treatment. Patients with a below-median proportion of EGFR-overlapping eccDNA at the response time point had dramatically better outcomes: progression-free survival of 803 vs 336 days (hazard ratio 0.24, 95% CI 0.09 to 0.61, p = 0.002) and overall survival of 1302 vs 476 days (hazard ratio 0.23, 95% CI 0.09 to 0.57, p less than 0.001).
Baseline Level Not Predictive. Importantly, the level of EGFR-overlapping eccDNA at baseline (before treatment) was not associated with survival outcomes. This indicates that the prognostic value of EGFR-overlapping eccDNA emerges during treatment, reflecting tumor response dynamics rather than pre-treatment tumor biology.
Why EGFR eccDNA During Treatment Matters. The finding that high EGFR-overlapping eccDNA during treatment predicts poor survival is biologically plausible: if effective osimertinib treatment kills cancer cells, fewer cells would be releasing EGFR-containing eccDNA. Persistent or increasing EGFR eccDNA may indicate inadequate tumor cell kill or emerging resistance.
eccDNA and Resistance Mechanisms. Prior research by Nathanson et al. proposed that disappearance of mutant EGFR from eccDNA can actually be a resistance mechanism -- cancer cells lose their dependence on EGFR when they develop other survival pathways. This may partly explain why some patients with lower EGFR-overlapping eccDNA still do well: it could reflect both genuine treatment efficacy and the absence of EGFR amplification-driven resistance.
Why Only Small eccDNA in Plasma. In contrast to tissue and cell line studies that detect eccDNA ranging from kilobases to megabases, plasma eccDNA was almost entirely below 2000 bp. The authors speculate this reflects either greater instability of large eccDNA in plasma (enzymatic degradation) or fragmentation of larger tissue eccDNA before its release into the bloodstream. The mechanism of eccDNA release (apoptosis, necrosis, or active secretion) remains incompletely understood.
Microhomology-Mediated Formation. Between 9.4% and 28.8% of eccDNA breakpoints showed microhomology (short direct repeat sequences of 2 to 15 bp), suggesting that microhomology-mediated end-joining contributes to eccDNA formation. However, the wide interpatient variation indicates this is not the only mechanism -- non-homologous end-joining and homologous recombination also likely play roles.
A New Liquid Biopsy Biomarker. This study establishes plasma eccDNA as a newly identified class of liquid biopsy biomarker for monitoring osimertinib treatment efficacy in EGFR-mutated NSCLC. Unlike circulating tumor DNA (ctDNA) that detects specific mutations, eccDNA analysis captures structural genomic alterations and oncogene amplification events.
Monitoring During Treatment. The clinical value of eccDNA as a biomarker lies specifically in on-treatment monitoring, not pre-treatment prediction. A simple blood draw after 4 weeks of osimertinib treatment could potentially identify patients unlikely to respond well, enabling earlier switch to alternative therapies or clinical trial enrollment.
Study Limitations. The primary limitation is the sample size of 32 patients, which restricts subgroup analyses and statistical power. Despite this, the study represents one of the largest homogeneous cohorts for plasma eccDNA research and is the first to prospectively examine eccDNA dynamics during systemic cancer treatment.
Broader Implications. While conducted in NSCLC, the findings may apply to eccDNA monitoring in other cancer types and EGFR-targeted therapies. Future prospective studies with larger cohorts are needed to validate eccDNA as a clinical biomarker and to explore its potential role in detecting emerging drug resistance before radiological progression becomes apparent.