| 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.Analysis 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 tumor tissues might be shorter than those derived from nonmalignant cells. For example, it has been shown that the proportion of DNA molecules carrying cancer-associated mutations would be higher when those mutations were detected using PCR with shorter amplicons (14, 27).In this study, we aimed to reconcile these apparent inconsistencies through the use of a study design that takes advantage of the following: (i) genome-wide high-resolution size profiling of plasma DNA enabled by massively parallel sequencing (28, 29); and (ii) an efficient approach to distinguish tumor-derived DNA from the nontumoral background DNA in the plasma of cancer patients. We believe that enhanced characterization of plasma DNA molecules in cancer patients would be useful for understanding the mechanisms involved in their generation and would offer useful insights for the development of diagnostic approaches.It has become feasible to measure the lengths of every individual plasma DNA molecule in samples with the use of massively parallel sequencing (28, 29). Hence, plasma DNA sizes could be studied in a genome-wide manner and at single-base resolution. Using this approach, the size of circulating DNA has generally been shown to resemble the size of mononucleosomal DNA, suggesting that plasma DNA might be generated through apoptosis (28, 29). In pregnant women, plasma DNA derived from the fetus has been shown to be shorter than that of DNA derived from the mother (28). The size difference between circulating fetal and maternal DNA has provided a previously unidentified conceptual basis for quantifying fetal DNA in maternal plasma and detecting chromosomal aneuploidies through size analysis of plasma DNA (30). In addition, differences in the size distributions of circulating DNA derived from the transplanted organs and the patients’ own tissues have been observed for recipients of solid organ or bone marrow transplantation (29).In this study, we used hepatocellular carcinoma (HCC) as a model to study the size distribution of plasma DNA in cancer patients. The size distributions of plasma DNA in HCC patients, patients with chronic hepatitis B virus (HBV) infection, patients with liver cirrhosis, and healthy subjects were also analyzed. Plasma of cancer patients contains a mixture of tumor-derived and non–tumor-derived DNA. We were particularly interested in studying the size profile of tumor-derived DNA in the plasma of the HCC patients. However, this is a challenging endeavor because tumor-derived plasma DNA could not be readily distinguished from the non–tumor-derived background DNA in plasma. The detection of cancer-specific mutations offers a genotypic means to distinguish the tumoral from the nontumoral plasma DNA. However, there are relatively few cancer-specific mutations across the genome (31–34) for the purpose of generating a broad, detailed, and yet cost-effective view of the size distribution of tumor-derived DNA.To circumvent this issue, we used chromosome arms that are affected by copy number aberrations (CNAs) to infer the difference in size distributions of tumor-derived and non–tumor-derived plasma DNA. The principle of this method is illustrated in . For chromosome arms that are amplified in the tumor tissues, the proportional contribution from tumor-derived DNA to plasma DNA would increase, whereas for chromosome arms that are deleted in the tumor, the contribution would decrease. Therefore, the comparison of size profiles of chromosome arms that are amplified and deleted would reflect the size difference between tumor-derived and non–tumor-derived DNA in plasma. CNA involving a whole chromosome arm or a large trunk of a chromosome arm is relatively common (35). Deletion of chromosomes 1p and 8p and amplification of chromosomes 1q and 8q are commonly observed in the HCC tissues (36–38). Thus, in this study, we focused on chromosomes 1 and 8 for the CNA and size-profiling analyses of plasma DNA. As the characteristic size profile of plasma nuclear DNA is likely to be related to histone packing, we hypothesized that the lack of histones packing for mitochondrial DNA might affect its abundance and size profile in plasma. Thus, we have also studied the size and fractional concentration of plasma mitochondrial DNA in the same cohort of subjects.Open in a separate windowSchematic illustration of the principle of plasma DNA size analysis in cancer patients. In cancer patients, plasma DNA is derived from both tumor (red molecules) and nontumor cells (blue molecules). Genomic regions that are amplified in the tumor tissue would contribute more tumoral DNA to plasma. Genomic regions that are deleted in the tumor tissue would contribute less DNA to plasma. Chromosome arm-level z-score analysis (CAZA) was used to determine if a chromosome arm is overrepresented or underrepresented in plasma DNA, suggestive of the presence of amplification or deletion, respectively, of the chromosome arm in the tumor. The size profiles of plasma DNA molecules originating from chromosome arms that are underrepresented (enriched for nontumor DNA) and overrepresented (enriched for tumor-derived DNA) were compared. |
