Genome-wide mapping of 5-hydroxymethylcytosines in circulating cell-free DNA as a non-invasive approach for early detection of hepatocellular carcinoma

Abstract

ObjectiveThe lack of highly sensitive and specific diagnostic biomarkers is a major contributor to the poor outcomes of patients with hepatocellular carcinoma (HCC). We sought to develop a non-invasive diagnostic approach using circulating cell-free DNA (cfDNA) for the early detection of HCC.DesignApplying the 5hmC-Seal technique, we obtained genome-wide 5-hydroxymethylcytosines (5hmC) in cfDNA samples from 2554 Chinese subjects: 1204 patients with HCC, 392 patients with chronic hepatitis B virus infection (CHB) or liver cirrhosis (LC) and 958 healthy individuals and patients with benign liver lesions. A diagnostic model for early HCC was developed through case-control analyses using the elastic net regularisation for feature selection.ResultsThe 5hmC-Seal data from patients with HCC showed a genome-wide distribution enriched with liver-derived enhancer marks. We developed a 32-gene diagnostic model that accurately distinguished early HCC (stage 0/A) based on the Barcelona Clinic Liver Cancer staging system from non-HCC (validation set: area under curve (AUC)=88.4%; (95% CI 85.8% to 91.1%)), showing superior performance over α-fetoprotein (AFP). Besides detecting patients with early stage or small tumours (eg, ≤2.0 cm) from non-HCC, the 5hmC model showed high capacity for distinguishing early HCC from high risk subjects with CHB or LC history (validation set: AUC=84.6%; (95% CI 80.6% to 88.7%)), also significantly outperforming AFP. Furthermore, the 5hmC diagnostic model appeared to be independent from potential confounders (eg, smoking/alcohol intake history).ConclusionWe have developed and validated a non-invasive approach with clinical application potential for the early detection of HCC that are still surgically resectable in high risk individuals.

<p>Dynamic Changes in Serum Markers and Their Utility in the Early Diagnosis of All Stages of Hepatitis B-Associated Hepatocellular Carcinoma</p>

“…Thus, specificity is affected to some extent. Therefore, nucleic acid markers are now largely developed towards based on their biology, such as methylation 35,36 and mutation, [37][38][39] to provide insight into treatment choice or potential drug resistance. As complete cells, CTCs can provide all the same information as nucleic acids.…”

Using circulating tumor DNA as a novel biomarker to screen and diagnose hepatocellular carcinoma: A systematic review and meta‐analysis

“…Additionally, the fluctuation in AFP levels is also associated with inflammation and liver disease type . For example, AFP levels may be enhanced in non‐HCC conditions, including chronic liver diseases (such as liver cirrhosis and hepatic inflammation), other tumors (such as intrahepatic cholangiocarcinoma and metastatic colon cancer) as well as pregnancy . Therefore, the detection of HCC with these methods remains suboptimal, it is imperative to develop additional biomarkers for early detection and diagnosis of HCC in a minimally invasive, convenient and accurate manner.…”

“…This assay, in conjunction with circulating tumor cells and circulating cell‐free DNA, is termed “liquid biopsy” . Circulating tumor DNA is generally derived from apoptotic or necrotic tumor cells and further released into the circulation, which carries cancer‐specific modifications in gene or epigenetics, including single nucleotide mutation, copy number aberration (CNA), and DNA methylation or 5‐hydroxymethylcytosines . Quantitative alteration and qualitative alteration of ctDNA are primarily detected in HCC patients.…”

“…18 Circulating tumor DNA is generally derived from apoptotic or necrotic tumor cells and further released into the circulation, 19 which carries cancer-specific modifications in gene or epigenetics, including single nucleotide mutation, 20 copy number aberration (CNA), 21 and DNA methylation 19 or 5-hydroxymethylcytosines. 16 Quantitative alteration and qualitative alteration of ctDNA are primarily detected in HCC patients. The former is associated with measuring the quantity of ctDNA that is generally increased in HCC patients, 22 and the latter refers to monitoring tumor-specific genetic aberrations.…”

5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic and predictive biomarkers for coronary artery disease

“…it has the potential to be biomarkers in different cancer types [53]. More recently, a diagnostic model based on features from cfDNA 5hmC in the blood showed the potential for early detection of hepatocellular carcinoma [54]. So the signal features of cfDNA 5hmC in the blood could be reliable biomarkers for different diseases.…”