Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients

Abstract

The analysis of tumor-derived circulating cell-free DNA opens up new possibilities for performing liquid biopsies for the assessment of solid tumors. Although its clinical potential has been increasingly recognized, many aspects of the biological characteristics of tumor-derived cell-free DNA remain unclear. With respect to the size profile of such plasma DNA molecules, a number of studies reported the finding of increased integrity of tumor-derived plasma DNA, whereas others found evidence to suggest that plasma DNA molecules released by tumors might be shorter. Here, we performed a detailed analysis of the size profiles of plasma DNA in 90 patients with hepatocellular carcinoma, 67 with chronic hepatitis B, 36 with hepatitis B-associated cirrhosis, and 32 healthy controls. We used massively parallel sequencing to achieve plasma DNA size measurement at single-base resolution and in a genome-wide manner. Tumor-derived plasma DNA molecules were further identified with the use of chromosome arm-level z-score analysis (CAZA), which facilitated the studying of their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of plasma mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These results have improved our understanding of the size profile of tumor-derived circulating cell-free DNA and might further enhance our ability to use plasma DNA as a molecular diagnostic tool.tumor markers | circulating tumor DNA | liquid biopsy | massively parallel sequencing | mitochondrial DNA A nalysis of circulating cell-free DNA has been increasingly used for the detection and monitoring of cancers (1-5). Different cancer-associated molecular characteristics, including copy number aberrations (6-9), methylation changes (10-13), single-nucleotide mutations (6, 14-17), cancer-derived viral sequences (18,19), and chromosomal rearrangements (20, 21), can be detected in the plasma of patients with various types of cancers. Despite the rapid expansion of clinical applications, many fundamental molecular characteristics of circulating DNA in cancer patients remain unclear. In particular, previous studies on the size of circulating DNA in cancer patients gave inconsistent results. Studies have demonstrated that the overall integrity of circulating DNA would increase in cancer patients compared with subjects without a malignant condition (22-25). Using PCR with different amplicon sizes, it was shown that the proportion of longer DNA would be higher in cancer patients. This aberration in DNA integrity was shown to be reversible after treatment, and the persistence of such changes was associated with poor prognosis (22, 26). On the other hand, there is also seemingly contradictory evidence that circulating DNA derived from t...

Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies

“…Assuming sufficiently high ligation efficiency, this would result in a higher fraction of mutant ctDNA molecules making it through sequencing pipeline than PCRbased applications. This assumption has been experimentally validated in libraries prepared from cfDNA of hepatocellular carcinoma patients (79). The authors observed a shift towards the characteristic cfDNA fragment length in read pairs mapping in regions associated with copy number gains, where we would expect greater proportion of ctDNA-derived fragments.…”

Novel insights into Notum and glypicans regulation in colorectal cancer

“…In this view, the analysis of the genomic instability resulting in copy number aberrations through massive parallel sequencing, as already demonstrated for prostate cancer [11] or the detection of tumorderived circulating cell-free DNA in plasma samples [12] could represent new possibilities for the detection and monitoring of CRC to investigate in the AOM/DSS murine model.…”

Potentials, challenges and limitations of a molecular characterization of circulating tumor DNA for the management of cancer patients

“…In order to accurately evaluate fragment sizes in cancer patients the Lo group made use of aneuploid regions of the cancer genome, in which ctDNA is either enriched as for amplified regions or depleted as for deleted regions [37]. Massively parallel sequencing revealed in addition to the 166 bp peak a series of smaller peaks occurring at 10 bp periodicity at sizes of approximately 143 bp and shorter [37]. Size profiles were highly correlated with the amount of tumor DNA in the circulation, whereas the abundance of shorter fragments was associated with larger amounts of tumor DNA [37].…”

“…Massively parallel sequencing revealed in addition to the 166 bp peak a series of smaller peaks occurring at 10 bp periodicity at sizes of approximately 143 bp and shorter [37]. Size profiles were highly correlated with the amount of tumor DNA in the circulation, whereas the abundance of shorter fragments was associated with larger amounts of tumor DNA [37]. Similar observations were already made in 2011, when the Thierry group demonstrated the presence of a higher proportion of cfDNA fragments below 100 bp particularly in samples from cancer [38].…”