Genome-scale detection of hypermethylated CpG islands in circulating cell-free DNA of hepatocellular carcinoma patients

Abstract

Despite advances in DNA methylome analyses of cells and tissues, current techniques for genome-scale profiling of DNA methylation in circulating cell-free DNA (ccfDNA) remain limited. Here we describe a methylated CpG tandems amplification and sequencing (MCTA-Seq) method that can detect thousands of hypermethylated CpG islands simultaneously in ccfDNA. This highly sensitive technique can work with genomic DNA as little as 7.5 pg, which is equivalent to 2.5 copies of the haploid genome. We have analyzed a cohort of tissue and plasma samples (n = 151) of hepatocellular carcinoma (HCC) patients and control subjects, identifying dozens of high-performance markers in blood for detecting small HCC (≤ 3 cm). Among these markers, 4 (RGS10, ST8SIA6, RUNX2 and VIM) are mostly specific for cancer detection, while the other 15, classified as a novel set, are already hypermethylated in the normal liver tissues. Two corresponding classifiers have been established, combination of which achieves a sensitivity of 94% with a specificity of 89% for the plasma samples from HCC patients (n = 36) and control subjects including cirrhosis patients (n = 17) and normal individuals (n = 38). Notably, all 15 alpha-fetoprotein-negative HCC patients were successfully identified. Comparison between matched plasma and tissue samples indicates that both the cancer and noncancerous tissues contribute to elevation of the methylation markers in plasma. MCTA-Seq will facilitate the development of ccfDNA methylation biomarkers and contribute to the improvement of cancer detection in a clinical setting.

Methylation-mediated repression of microRNA-129-2 suppresses cell aggressiveness by inhibiting high mobility group box 1 in human hepatocellular carcinoma

“…Several studies revealed that promoter hypermethylation induced downregulation of miRNAs closely correlates with carcinogenesis [42]. Our data demonstrated that hypermethylation of upstream of miR-129-2 led to the downregulation of miR-129-2 in both HCC tissues and cell lines.…”

Circulating Tumor DNA as Biomarkers for Cancer Detection

“…Given ctDNA is fragmented and accounts for only a small portion of cfDNA, this sensitive method is suitable for detecting ctDNA methylation, since it only requires small amounts of ctDNA (as low as 7.5 pg) [173]. This is the first genome-wide technique developed for the detection of ctDNA methylations (Table 4).…”

Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper‑tract urothelial carcinoma

“…VIM methylation has been found to be more frequent in bladder urothelial carcinoma and UTUC, but rare in normal tissue, and may therefore be useful as a novel diagnosis and detection method in urothelial cancer. Downregulation of VIM has also been associated with increased tumor invasion, progression, epithelial to mesenchymal transition (EMT) and poor prognosis in various types of tumor (30)(31)(32)(33)(34). Monteiro-Reis et al (31) suggested that during early upper urinary tract carcinogenesis, the VIM promoter is progressively methylated and the gene is kept silenced, as in normal urothelium; by contrast, in a subset of UTUCs, methylation is decreased, allowing for aberrant vimentin expression, due to stimuli leading to EMT.…”