Schematic 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. 

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... 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 un- identified 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 be- cause tumor-derived plasma DNA could not be readily distin- guished from the non – tumor-derived background DNA in plasma. The detection of cancer-specific mutations offers a ge- notypic 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 gener- ating 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 Fig. 1. 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. Detection. We analyzed a total of 225 plasma DNA samples from 90 HCC patients, 67 patients with chronic HBV infection, 36 patients with HBV-associated liver cirrhosis, and 32 healthy subjects. For the HCC patients, 85 (94.4%) had Barcelona Clinic Liver Cancer stage A disease and 5 (5.6%) had stage B disease. A median of 31 million reads (range: 17 – 79 million) was obtained from each plasma sample. Amounts of sequence reads originating from chromosome arms that were 3 SDs below ( z scores less than − 3) and 3 SDs above ( z scores greater than 3) the mean of healthy controls were deemed to indicate significant underrepresentations and overrepresentations of the plasma DNA from those chromosome arms, respectively. These plasma DNA quantitative aberrations were generally reflective of the presence of copy number losses and copy number gains (CNAs) in the tumor (6) (Fig. S1). The plasma CNA results by chromosome arm-level z -score analysis (CAZA) of chromosomes 1 and 8 for all of the HCC patients and controls are shown in Fig. 2 and summarized in Table 1. Seventy-six (84.4%) of the 90 HCC patients had at least one chromosomal arm-level CNA on chromosomes 1 and 8 in plasma. Tumor tissues of 12 HCC patients were available to corrob- orate the plasma DNA findings. The tissue samples were sequenced, and the CNA patterns are shown in Fig. 3. Of the 48 chromosome arms analyzed for the 12 patients, concordant changes in plasma and tumor tissues were observed for 30 (63%) arms. The total plasma DNA concentrations were measured using quantitative real-time PCR targeting the HBB (hemoglo- bin, beta) gene (39). CNAs were only observed in the tumors but not in the plasma for 10 (21%) arms. These cases tended to have lower tumor DNA fractions in the plasma. CNAs were observed in the plasma but not the tumors for 7 (15%) arms. In one case (HOT428), gain of 1q was observed in the tumor, but a loss was observed in the plasma. These data might reflect the presence of tumoral heterogeneity where there might be other foci or clones of cancer cells contributing plasma DNA. Among the HBV carriers with and without liver cirrhosis, the detection rates of these CNA were 22.2% and 4.5%, respectively. Interestingly, one HBV carrier with cirrhosis and another one without cirrhosis exhibited CNA in the plasma but, not known to have HCC at the time of blood collection, were diagnosed with HCC at 3 and 4 mo afterward, respectively. All of the HBV carriers and cirrhotic patients were followed up for at least 6 mo. For those control subjects without any CNA in plasma, none of them had developed HCC during the follow-up period. The clinical significance of having CNA in plasma for subjects without a current cancer would warrant further investigation with longer follow-up of these subjects. None of the 32 healthy subjects had detectable CNA on chromosome 1 or 8 in plasma by CAZA. In the HCC patients, the disproportionate increase or decrease in sequence reads in the plasma due to the presence of CNAs is reflective of the fractional concentrations of tumor DNA in the plasma samples. The median fractional concentration of tumor-derived DNA in the plasma of the HCC patients was 2.1% (range: 0 – 53.1%; interquartile range: 1.2 – 3.8%). Plasma DNA Size Distribution of HCC Patients. The size distributions of plasma DNA of the HCC patients, HBV carriers with and without cirrhosis, and healthy controls are shown in Fig. 4 and Fig. S2. In general, the most prominent peak was observed at 166 bp in the size distribution plot of each subject. This observation is consistent with previous reports on pregnant women and transplant recipients (28 – 30), suggesting that most of the circulating DNA molecules are derived from apoptosis. Interestingly, compared with healthy controls (black line in Fig. 4), the sizes of plasma DNA in HCC patients with low ...