Impact of Long-Term Plasma Storage on Cell-Free DNA Epigenetic Biomarker Studies

Abstract

Impact of long-term plasma storage on biomarker analysis is critical for ensuring data reliability. Cell-free DNA (cfDNA) epigenetic markers, including 5-hydroxymethylcytosine (5hmC), have emerged for disease detection, prognosis, and treatment response. However, the effects of prolonged storage on 5hmC analysis remain unclear. We evaluated the quantity and quality of cfDNA and 5hmC sequencing analyses in 1070 plasma samples stored for up to 14 years from patients with solid tumors and acute myeloid leukemia (AML) and non-cancer individuals. In long-term stored plasma samples, cfDNA yield remained largely stable; however, uniquely mapped reads (UMRs) from 5hmC sequencing were significantly reduced in solid tumor and control samples. Notably, prolonged plasma storage independently contributed to increased genomic DNA (gDNA) contamination in solid tumor and AML samples and significantly correlated with decreased UMRs in control samples. Across all groups, samples with gDNA contamination exhibited significantly reduced UMRs. Furthermore, gDNA contamination independently compromised cfDNA fragment integrity, decreased sequencing library success in solid tumors, and reduced 5hmC sequencing UMRs across all groups. Therefore, extended plasma storage contributes to increased gDNA contamination, compromising cfDNA and 5hmC sequencing quality. Implementing measures to minimize gDNA contamination in long-term plasma storage is crucial for improving downstream cfDNA analysis reliability.

Keywords: 5-hydroxymethylcytosine; cancer; cell-free DNA; genomic DNA; plasma storage.

Figure 1

Cell-free DNA (cfDNA) yield in long-term stored plasma samples. (A) cfDNA yield in plasma samples from patients with solid tumors and acute myeloid leukemia (AML) and non-cancer controls. (B) cfDNA yield in plasma samples from patients with AML and controls based on plasma storage time. (C) cfDNA yield in plasma samples from patients with solid tumors based on plasma storage time. (D) cfDNA yield of plasma samples based on genomic DNA (gDNA) contamination. (E) cfDNA yield of plasma samples from patients with solid tumors based on elapsed processing time. (F) cfDNA yield of plasma samples from patients with AML based on elapsed processing time. (G) cfDNA yield of plasma samples from patients with solid tumors and AML and controls based on sex. (H) cfDNA yield of plasma samples based on age. (I) cfDNA yield of plasma samples from patients with solid tumors based on cancer stages. Kruskal–Wallis test or Wilcoxon Rank Sum test was performed. Bounds of box represent 25th and 75th percentiles and whiskers are Tukey whiskers. p values < 0.05 are indicated.

Figure 2

Uniquely mapped reads (UMRs) in 5hmC sequencing analysis. (A) UMRs of plasma samples from patients with AML and controls based on plasma storage time. (B). UMRs of plasma samples from patients with solid tumors based on plasma storage time. (C). UMRs of plasma samples based on gDNA contamination. (D). UMRs of plasma samples based on sex. (E) UMRs of plasma samples based on age. (F). UMRs of plasma samples from patients with solid tumors based on cancer clinical stages. (G) UMR yield of plasma samples from patients with solid tumors and AML based on elapsed processing time. Kruskal–Wallis test or Wilcoxon Rank Sum test was performed. Bounds of box represent 25th and 75th percentiles and whiskers are Tukey whiskers. p values < 0.05 are indicated.