Cell-free DNA copy number variations in plasma from colorectal cancer patients

Abstract

To evaluate the clinical utility of cell-free DNA (cfDNA), we performed whole-genome sequencing to systematically examine plasma cfDNA copy number variations (CNVs) in a cohort of patients with colorectal cancer (CRC, n = 80), polyps (n = 20), and healthy controls (n = 35). We initially compared cfDNA yield in 20 paired serum-plasma samples and observed significantly higher cfDNA concentration in serum (median = 81.20 ng, range 7.18-500 ng·mL^-1 ) than in plasma (median = 5.09 ng, range 3.76-62.8 ng·mL^-1 ) (P < 0.0001). However, tumor-derived cfDNA content was significantly lower in serum than in matched plasma samples tested. With ~10 million reads per sample, the sequencing-based copy number analysis showed common CNVs in multiple chromosomal regions, including amplifications on 1q, 8q, and 5q and deletions on 1p, 4q, 8p, 17p, 18q, and 22q. Copy number changes were also evident in genes critical to the cell cycle, DNA repair, and WNT signaling pathways. To evaluate whether cumulative copy number changes were associated with tumor stages, we calculated plasma genomic abnormality in colon cancer (PGA-C) score by summing the most significant CNVs. The PGA-C score showed predictive performance with an area under the curve from 0.54 to 0.84 for CRC stages I-IV. Locus-specific copy number analysis identified nine genomic regions where CNVs were significantly associated with survival in stage III-IV CRC patients. A multivariate model using six of nine genomic regions demonstrated a significant association of high-risk score with shorter survival (HR = 5.33, 95% CI = 6.76-94.44, P < 0.0001). Our study demonstrates the importance of using plasma (rather than serum) to test tumor-related genomic variations. Plasma cfDNA-based tests can capture tumor-specific genetic changes and may provide a measurable classifier for assessing clinical outcomes in advanced CRC patients.

Keywords: cell-free DNA; colon cancer; copy number variation; next-generation sequencing; survival.

Figure 1

Comparison of copy number changes between four pairs of serum and plasma. (A) Heatmap of log2 ratio in 1‐Mb genomic window shows higher tumor‐specific cfDNA in plasma than in serum. Red color represents copy number gain, while blue represents loss. Intensity of the color is proportional to the value of log2 ratio and reflects the weight of tumor‐specific cfDNA in overall background cfDNA. (B) Segmentation‐based copy number analysis shows more prominent copy number changes in plasma than in serum. Most significant segment losses (arrows) were used to calculate tumor‐specific cfDNA difference between serum and plasma.

Figure 2

Association of PGA‐C score with disease status. (A) PGA‐C score increases with disease progression. (B) AUC analysis shows predictive performance of distinguishing CRC cases from healthy controls.

Figure 3

Representative gene regions with copy number changes. Each copy number change is illustrated in overall genomic view (upper panel) and detailed gene region view (lower panel).

Figure 4

Oncoprint of copy number changes in critical genes of CRC‐related pathways. Red and blue represent gain and loss, respectively. Common CNVs are defined as ≥3 gains or losses (but not both) in all 79 cases tested.

Figure 5

Kaplan–Meier analysis of overall survival. Six genomic segment‐based predictive model defines high‐risk group with median survival = 15.87 months and low‐risk group with median survival = 68.53 months in stage III‐IV CRC patients.