GSE63775
MCTA-Seq for profiling hypermethylated CpG islands in plasma of hepatocellular carcinoma patients
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. Overall design: First, for validation of the MCTA-Seq techique, we examined CpG hypermethylation in fully methylated genomic DNA, and the genomic DNAs of WBC and 2 cancer cell lines. Then, we performed the MCTA-Seq method to clinical samples, including 27 pairs of HCCs and matching adjacent noncancerous liver samples and 3 normal liver samples, as well as plasma cell-free DNAs obtained from 27 HCC patients, 16 cirrhosis patient and 29 healthy individuals as the training group, and plasma cell-free DNAs obtained from 9 HCC patients and 10 healthy individuals as the testing group.