Delineating the structural features of circulating DNA by unraveling the puzzle their fragment size distribution

We scrutinized the cfDNA size distribution in order to delineate their structures in blood of healthy individuals and cancer patients. This exploratory study was based on the blinded examination of four plasma from healthy individuals and the plasma of four metastatic colorectal cancer (mCRC) patients presenting a wide variation of mutant allele frequency (1.5%, 7.1%, 25.7% and 79.4%). CfDNA size distribution in these healthy and mCRC subject plasma was determined by using whole genome sequencing from both double- and single-strand DNA library preparations as well as by Q-PCR. CfDNA integrity was assessed in “ad hoc” study from 117 healthy individuals and 112 CRC patients. The size profile below 1,000 bp in healthy individuals is remarkably homogenous among the tested plasma in contrast to that of CRC plasma cfDNA which proportionally varied with the mutant allele frequency. When combining data from the three analytical approaches and both cohorts, we estimated that the proportion cfDNA inserted in mono-nucleosomes, di-nucleosomes and of higher molecular size (>1000bp) are 87.5%, 1.1% and 11.4% in plasma of healthy individuals, and are 93.5-97.8%, 2.1% and 0.1-4.4% in plasma of CRC patient plasma, respectively. Thus, our data suggested that the proportion of cfDNA having a size over that existing in mono-nucleosome or Transcription Factor complexes circulating in blood of either healthy or cancer individuals is minor, suggesting that cfDNA which can be detected in plasma is predominantly associated by both structures. Although the general size distribution of cfDNA from healthy and cancer individuals is similar, our data revealed subtle but reliable differences in the 30-220 bp range, and to a lesser extent in cfDNA higher of about 1,000bp. Our study showed that detection of cfDNA from cancer cells is possible by examining cfDNA size profile and calculating the proportion of the number of fragments in specific size ranges.