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.

Keywords: circulating tumor DNA; liquid biopsy; massively parallel sequencing; mitochondrial DNA; tumor markers.

Conflict of interest statement

Conflict of interest statement: R.W.K.C. and Y.M.D.L. received research support from Sequenom, Inc. R.W.K.C. and Y.M.D.L. are consultants to Sequenom, Inc. R.W.K.C., K.C.A.C., and Y.M.D.L. hold equities in Sequenom, Inc. R.W.K.C., K.C.A.C., and Y.M.D.L. are founders of Xcelom. P.J., R.W.K.C., K.C.A.C., and Y.M.D.L. have filed patents/patent applications based on the data generated from this work.

Figures

Fig. 1.

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.

Fig. 2.

Plasma CNA results for all studied subjects by CAZA. The four chromosome arms (1p, 1q, 8p, and 8q) that are frequently affected by CNAs in HCC were analyzed. Red and green lines represent underrepresentation and overrepresentation, respectively, of the corresponding chromosome arms in plasma. Each vertical line represents the data for one case. The vertical arrows at the bottom of the figure indicate the two cases in which HCC was diagnosed 3–4 mo following blood sampling.

Fig. 3.

CNAs detected in the tumor and corresponding plasma of 12 HCC patients. The patients are arranged in descending order of tumor DNA fraction in plasma. A total of 48 chromosome arms were analyzed for the 12 patients. The numbers (and percentages) of chromosome arms with concordant and discordant results between tumor and plasma are shown.

Fig. 4.

Size distributions of plasma DNA fragments in the HCC patients with different fractional concentrations of tumor-derived DNA in plasma. The median size distribution profile for the 32 healthy subjects is shown as a thick black line.

Fig. 5.

Plots of the proportions of plasma DNA fragments of (A) shorter than 150 bp, (B) from 150 to 180 bp, and (C) longer than 180 bp against tumor DNA fraction in plasma. The tumor DNA fraction in plasma is shown in logarithmic scale.

Fig. 6.

Size distributions of plasma DNA originating from the amplified 8q and deleted 8p of an illustrative case (H291). (A) The size distributions of plasma DNA for 8p (red) and 8q (green). (B) Plot of cumulative frequencies for plasma DNA size for 8p (red) and 8q (green). (C) The difference in cumulative frequencies, denoted as ∆S, between 8q and 8p for the HCC case H291 (red line). Gray lines are the results for all healthy control subjects.

Fig. 7.

The difference in the cumulative frequencies for size between 8q and 8p (∆S). (A) Plot of ∆S against size for all of the HCC cases with different CNAs on 8p and 8q in plasma. Cases with different ranges of fractional tumor DNA concentrations in plasma are shown in different colors. (B) The values of ∆S166 among different groups. For the HCC group, patients with and without different CNAs on 8p and 8q as determined by plasma CAZA analysis are represented by red and black dots, respectively.

Fig. 8.

(A) The fractional concentration of mitochondrial DNA in plasma. (B) ROC curve on the use of fractional concentration of mitochondrial DNA in plasma for discriminating the HCC patients from the healthy subjects.

Fig. 9.

The size profiles of circulating mitochondrial DNA in healthy subjects (black), HBV carriers (yellow), cirrhotic patients (blue), and HCC patients (red). The size profile of circulating nuclear DNA of one healthy control subject is shown for comparison (dotted line).