Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers

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Most cancer researchers, as well as physicians and patients, are surprised when they discover that circular extrachromosomal DNA (ecDNA) is a major driver of cancer pathogenesis. “Why didn’t I know about this?” This query is usually followed by further questions - how common is ecDNA? In which cancer types does ecDNA occur? How do you detect it? Do you see it in normal tissue? Does it impact prognosis? Our new paper in Nature Genetics answers these critical questions.

It seems that cancers have pulled an ancient evolutionary trick. Oncogenes and surrounding regulatory regions untether themselves from their chromosomal constraints, driving high oncogene copy number, accelerating tumor evolution, contributing to therapeutic resistance, and endowing tumors with the ability to rapidly change their genomes in response to rapidly changing environments, thereby accelerating tumor evolution and contributing to therapeutic resistance1-4. ecDNAs are relatively large (1.3 Mb on average), highly amplified circular structures containing genes and regulatory regions. ecDNAs are wound around histone cores into nucleosomes, but in a highly abnormal fashion5. They contain an abundance of active chromatin marks and demonstrate a paucity of repressive chromatin. Further, there is a defect in the higher order organization of nucleosomes, making ecDNAs amongst the most accessible chromatin in the genome of tumor cells5. The unique circular topology adds an additional wrinkle, generating new, cis-regulatory interactions created by its circular shape. There are real consequences to these profound alteration of the epigenome on circular ecDNA, including massive oncogene transcription and potential regulatory re-wiring5, a finding that has recently been beautifully independently confirmed by other research teams6,7. These studies reveal an even deeper layer of complexity to cancer genomes than first thought, overturning current dogma of how some of the most aggressive cancer evolve, challenging existing “maps” of cancer genomes, and opening an entirely new field in cancer research. From a historical perspective, it is important to note that modern research into ecDNA shows how prescient the efforts of the earlier pioneering scientists who studied extrachromosomal amplification were, conducting their analyses long before the availability of powerful DNA sequencing technologies and the mapping of the human genome8-12. Indeed, it was no surprise that until very recently, ecDNA was thought to be a very rare phenomenon of unknown biological and clinical significance (1.4% of cancers, https://mitelmandatabase.isb-cgc.org/). No wonder very few cancer researchers “knew about it”.

This brings us back to the initial fundamental question, that has, until now, been very difficult to answer. How common is ecDNA in human cancer? How do we find that which has been hiding in plain sight? Enormous communal investments have been made in obtaining DNA sequencing information from thousands of cancer samples, providing remarkable power to identify genetic alterations and better understand their impact. But, can we use these publicly available data to determine the frequency and clinical impact of ecDNA? Whole exome sequencing is lacking critical information, as the exomes contain less the 2% of the total bulk of DNA. Fortunately, publicly available databases, including TCGA and PCAWG, contain a large number of cancer samples for which whole genome sequencing has been deposited, yielding a golden opportunity for discovery. We developed a powerful computational approach called Amplicon architect, which identifies ecDNA based on three key features– circularity, high copy number, and reuse of breakpoints. We applied Amplicon Architect to over 3200 cancer samples of a wide range of histological types, and matched whole blood and normal tissue, we were able to determine the frequency of ecDNA by cancer type, as well as deciphering other types of gene amplification events, and were then able to examine some highly relevant clinical and biological correlates of ecDNA driven cancers. Remarkably, we find that ecDNA is a common event in human cancer, at minimum 14% of human tumors, with far, far higher frequencies in the most malignant forms of cancer, including GBM, sarcoma, esophageal, ovarian, lung, bladder, head and neck, gastric, and many others.

Fig. 1 Frequency of circular amplification across tumor and non-tumor tissues (taken from Fig 1 of the paper). The distribution of circular ecDNA, and other amplification types including Breakage Fusion Bridges (BFB), Heavily-rearranged amplicons, Linear amplifications, and no focal somatic copy number amplifications detected (No-fSCNA), by tumor type across 3,731 tumor and matched non-neoplastic samples.


We further found that ecDNA amplification is massively enriched for highly amplified oncogenes, including EGFR, MDM2, MYC, CDK4, ERBB2, and many other bona fide oncogenes, on highly accessible chromatin with massively elevated transcription. Importantly, ecDNA-associated cancers appear to be immune “cold”. ecDNA is also a cancer-specific phenomenon, being virtually undetectable in matched normal tissue and blood. Finally, and most importantly, we find that patients whose cancers have ecDNA have significantly shorter survival than all other cancer patients, whose tumors are driven by other molecular lesions, even when stratified by tumor type.


Fig. 2 Presence of circular amplification associates with poor outcomes (taken from Fig. 4 of the paper). Kaplan-Meier five-year survival curves by amplification category. Patients whose tumors contain at least one Circular ecDNA-based amplicon have significantly worse outcome compared to patients whose tumors were classified as non-circular. The p-value comparing survival curves was based on a log-rank test.

 This study provides a new window into the molecular epidemiology of ecDNA in cancer, providing a unique opportunity to study patients longitudinally to better understand how and why they respond poorly to treatment. Our new study also poses a challenge to the precision oncology field. Patients whose cancers are driven by ecDNA are different. We look forward to collectively devising new therapeutic strategies and more effective treatments for these patients whose tumors harbor ecDNA. These patients have been poorly served by existing standards of care.



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                Paul Mischel, M.D.

                Professor, UCSD/Ludwig

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