Cell‐free DNA 5‐hydroxymethylcytosine as a marker for common cancer detection

Abstract

Background Early cancer detection can dramatically improve clinical outcomes and greatly reduce economic burden of patients. Plasma cell–free DNA (cfDNA) 5‐hydroxymethylcytosine (5hmC) is an emerging epigenetic marker for cancer diagnosis. However, the utility of such marker has not been investigated in many common cancers yet. The purpose of this study was to evaluate 5hmC in plasma cfDNA for an early detection of common cancers. Methods We used a highly sensitive nano‐5hmC‐Seal method and profiled the genome‐wide distribution of 5hmC in plasma cfDNA from 384 patients with bladder, breast, colorectal, kidney, lung or prostate cancer and 221 controls. Genes and signalling pathways with differential hydroxymethylation were analysed. We used machine learning to develop 5hmC signatures for cancer detection and cancer‐origin determination in the training sets and validated the signatures in the validation sets. Results We identified genes and signalling pathways with aberrant DNA hydroxymethylation in six cancers. We discovered a pan‐cancer signature that detected all six cancers with a sensitivity of 68.6% and a specificity of 96.6% and cancer–specific signatures with a sensitivity of 80.0% for breast cancer, 88.9% for kidney cancer, 94.1% for lung cancer and 96.4% for prostate cancer, and a specificity of 100% for all except lung (96.2%). The sensitivity of cancer–specific signatures was 89.3%–100.0% for early‐stage cancers. The lung cancer–specific signature achieved a sensitivity of 98.0% and a specificity of 82.3% in an independent patient cohort with different ethnic backgrounds. Additionally, we discovered a 5hmC signature that could accurately determine cancer origin. Conclusions We demonstrated that plasma cfDNA 5hmC is a highly sensitive biomarker for common cancer detection. Our genome‐wide analysis of 5hmC in six cancers reveals new target genes and signalling pathways with therapeutic potential for common cancers.

Cell-free DNA 5-hydroxymethylcytosine is highly sensitive for MRD assessment in acute myeloid leukemia

“…Plasma cfDNA extraction, library preparation, and NGS were performed as previously described [ 8 , 9 ]. High-quality reads were counted into gene bodies (RefSeq) using featureCounts.…”

“…A high level of 5hmC is associated with adverse OS in AML [ 7 ]. Using a highly sensitive nano-hmC-Seal method, we and others have demonstrated that plasma cell-free DNA (cfDNA) 5hmC is highly sensitive for the detection and prognosis of AML and other malignancies [ 5 , 6 , 8 , 9 ]. Thus, we hypothesized that cfDNA 5hmC could act as a highly sensitive marker of MRD in AML.…”

Classification of Acute Myeloid Leukemia by Cell-Free DNA 5-Hydroxymethylcytosine

“…We assessed cfDNA quantity and quality using the Qubit Fluorometer with dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and Bioanalyzer 2100 with Agilent High Sensitivity Assay Kit (Agilent Technologies, Santa Clara, CA, USA). We constructed the 5hmC library as previously described [ 21 , 22 ]. Briefly, we ligated cfDNA with adaptors before incubation with N3-UDP-azide-glucose and T4 Phage β-glucosyltransferase at 37 °C for 1 h. We then purified the DNA and incubated it with DBCO-PEG4-DBCO at 37 °C for 2 h. The DNA libraries were sequenced using the NextSeq 550 and NovaSeq 6000 instruments (Illumina, San Diego, CA, USA).…”

“…Briefly, we ligated cfDNA with adaptors before incubation with N3-UDP-azide-glucose and T4 Phage β-glucosyltransferase at 37 °C for 1 h. We then purified the DNA and incubated it with DBCO-PEG4-DBCO at 37 °C for 2 h. The DNA libraries were sequenced using the NextSeq 550 and NovaSeq 6000 instruments (Illumina, San Diego, CA, USA). Sequencing data were processed as previously described [ 21 , 22 ]. Briefly, we first evaluated the raw sequencing read quality with FastQC ( (accessed on 1 May 2020).…”

“…Our previous study demonstrated that the number of differentially hydroxymethylated genes with increased 5hmC levels exceeded those with decreased 5hmC levels and involved more signaling pathways in AML patients [ 21 ]. Because 5hmC is enriched primarily in distal regulatory regions, gene bodies, and promoters [ 2 , 3 , 20 , 21 , 22 ], the delineation of 5hmC distribution in specific genomic regions may reveal critical aspects of the biology underlying AML. To this end, we and others have shown that plasma cell-free DNA (cfDNA) 5hmC is an effective, minimally invasive, and highly sensitive marker for detection and prognosis in AML and multiple malignancies [ 2 , 3 , 21 , 22 ].…”