Normal and Cancerous Tissues Release Extrachromosomal Circular DNA (eccDNA) into the Circulation

Abstract

Cell-free circulating linear DNA is being explored for noninvasive diagnosis and management of tumors and fetuses, the so-called liquid biopsy. Previously, we observed the presence of small extrachromosomal circular DNA (eccDNA), called microDNA, in the nuclei of mammalian tissues and cell lines. Now, we demonstrate that cell-free microDNA derived from uniquely mapping regions of the genome is detectable in plasma and serum from both mice and humans and that they are significantly longer (30%-60% >250 bases) than cell-free circulating linear DNA (∼150 bases). Tumor-derived human microDNA is detected in the mouse circulation in a mouse xenograft model of human ovarian cancer. Comparing the microDNA from paired tumor and normal lung tissue specimens reveals that the tumors contain longer microDNA. Consistent with human cancers releasing microDNA into the circulation, serum and plasma samples (12 lung and 11 ovarian cancer) collected prior to surgery are enriched for longer cell-free microDNA compared with samples from the same patient obtained several weeks after surgical resection of the tumor. Thus, circular DNA in the circulation is a previously unexplored pool of nucleic acids that could complement miRNAs and linear DNA for diagnosis and for intercellular communication.Implications: eccDNA derived from chromosomal genomic sequence, first discovered in the nuclei of cells, are detected in the circulation, are longer than linear cell-free DNA, and are released from normal tissue and tumors into the circulation. Mol Cancer Res; 15(9); 1197-205. ©2017 AACR.

Figure 1. Properties of circulating microDNA from mouse serum are similar to those observed in mouse tissues

(A) Length distribution of microDNAs identified in the serum of two mice. (B) Median percent GC content of microDNAs and the genomic sequences of equal length upstream or downstream of the microDNA source loci are enriched relative to the average GC content of the mouse genome (dashed line). (C) Distribution of microDNA in the indicated genomic regions. (D) Percentage of microDNA with (black) or without (gray) 2- to 15-bp direct repeats flanking the microDNA locus at the genomic source.

Figure 2. Properties of human-derived microDNA in serum of mice with human ovarian cancer xenografts

(A) Length distribution of human microDNAs identified in the serum of two mice carrying xenografts of the human ovarian cancer SKOV3 cell line. (B) Median percent GC content of human-derived microDNAs and the genomic sequences of equal length upstream or downstream of the microDNA source locus are enriched relative to the average GC content of the human genome (dashed line). (C) Distribution of human-derived microDNA in the indicated genomic regions. (D) Percentage of microDNA with (black) or without (gray) 2- to 15-bp direct repeats flanking the microDNA sequence at the genomic source loci.

Figure 3. Cumulative distribution function (CDF) plot of microDNA lengths in lung tumor and matched normal tissue

(A–D) Plot showing the CDF of microDNA lengths in tumor (black line) and matched normal tissue (gray line) in each patient. The cumulative frequency was calculated by using ecdf function in R. The further to the right a plot is, the longer the microDNA in that sample. (E–H) Plot showing the CDF of microDNA in pre surgery (black line) and post surgery (gray line) plasma sample in each patient. (I–L) Plot showing the CDF of microDNA length in pre surgery (black line) and post surgery (gray line) serum sample in each patient. In all plots the statistical significance of the length difference in tumor vs. normal or pre surgery vs. post surgery sample was calculated by a one-sided k-s test (tumor microDNA plot is below normal microDNA plot; pre-surgery microDNA plot is below post-surgery microDNA plot) and the p-value indicated.

Figure 4. Cumulative distribution function (CDF) plot of length of microDNA isolated from plasma of lung cancer patients pre surgery and post surgery

(A–H) Plot showing the CDF of lengths of microDNA in pre surgery (black line) and post surgery (gray line) plasma sample in each patient. The rest as in Fig. 3. In five out of eight samples microDNA length in pre surgery plasma sample were significantly longer.

Figure 5. Cumulative length distribution (CDF) plot of microDNA isolated from plasma of ovarian cancer patients pre surgery and post surgery

(A–K) Plot showing the CDF of lengths of microDNA in pre surgery (black line) and post surgery (gray line) plasma sample in each patient. Rest as in Fig. 3. In seven out of eleven samples microDNA length in pre surgery plasma sample were significantly long.