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Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer.

ABSTRACT: Early detection of cancer will improve survival rates. The blood biomarker 5-hydroxymethylcytosine has been shown to discriminate cancer. In a large covariate-controlled study of over two thousand individual blood samples, we created, tested and explored the properties of a 5-hydroxymethylcytosine-based classifier to detect colorectal cancer (CRC). In an independent validation sample set, the classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (95% CI [87, 93]). Sensitivity was 55% at 95% specificity. Performance was similar for early stage 1 (AUC 89%; 95% CI [83, 94]) and late stage 4 CRC (AUC 94%; 95% CI [89, 98]). The classifier could detect CRC even when the proportion of tumor DNA in blood was undetectable by other methods. Expanding the classifier to include information about cell-free DNA fragment size and abundance across the genome led to gains in sensitivity (63% at 95% specificity), with similar overall performance (AUC 91%; 95% CI [89, 94]). We confirm that 5-hydroxymethylcytosine can be used to detect CRC, even in early-stage disease. Therefore, the inclusion of 5-hydroxymethylcytosine in multianalyte testing could improve sensitivity for the detection of early-stage cancer.

SUBMITTER: Walker NJ

PROVIDER: S-EPMC9532421 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer.

Walker Nicolas J NJ Rashid Mamunur M Yu Shirong S Bignell Helen H Lumby Casper K CK Livi Carmen M CM Howell Kate K Morley David J DJ Morganella Sandro S Barrell Daniel D Caim Shabhonam S Gosal Walraj W Füllgrabe Jens J Charlesworth Thomas J TJ Vasquez Louella L Ahdesmäki Miika M Eizenga Jordan J Prabhat Parul P Proutski Vitali V Murat-Onana Marie Laurie ML Greenwood Catherine J CJ Kirkwood Lisa L Maisuria-Armer Meeta M Li Mengjie M Coats Emma E Winfield Victoria V MacBean Lachlan L Stock Toby T Tomé-Fernandez Alice A Chan Yat Y Sheikh Nasir N Golder Paula P Steward Michael M Ost Tobias W B TWB Stewart Douglas D Vilella Albert A Noursalehi Mojtaba M Paten Benedict B Lucarelli Debora D Mason Joanne J Ridge Gareth G Mellad Jason J Shirodkar Suman S Balasubaramanian Shankar S Holbrook Joanna D JD

Scientific reports 20221004 1

Early detection of cancer will improve survival rates. The blood biomarker 5-hydroxymethylcytosine has been shown to discriminate cancer. In a large covariate-controlled study of over two thousand individual blood samples, we created, tested and explored the properties of a 5-hydroxymethylcytosine-based classifier to detect colorectal cancer (CRC). In an independent validation sample set, the classifier discriminated CRC samples from controls with an area under the receiver operating characteris ...[more]

PMID: 36195648

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Project description:BackgroundAs a crucial epigenetic modification, DNA 5-hydroxymethylcytosine (5-hmC) plays a key role during colorectal cancer (CRC) carcinogenesis. Nevertheless, the levels of 5-hmC-related genes in the circulating DNA of CRC remain largely unknown.Methods and resultsThe GSE81314 dataset from the Gene Expression Omnibus (GEO), which was generated by chemical marking-based low-input shotgun sequencing to detect 5-hmC in circulating cell-free DNA (cfDNA) was used in the present study. The GSE81314 dataset includes data for 8 plasma samples from healthy individuals and 4 plasma samples from CRC patients. The difference in the 5-hmC levels in cfDNA between the CRC group and healthy individuals was analyzed by the differentially expressed genes (DEG) package. Weighted gene coexpression network analysis (WGCNA) was conducted to analyze gene coexpression modules associated with sample characteristics. DEG analysis identified 19 upregulated and 9 downregulated 5-hmC-related genes. WGCNA showed that the pink, purple, and brown modules, which contain 531 genes in total, were significantly correlated with CRC (0.66, 0.61, and -0.59, respectively). We used gene set enrichment analysis (GSEA) software to compare 5-hmC-related genes and pathways between CRC patients and healthy controls. We further performed a protein-protein interaction (PPI) analysis and identified 4 nodes (LCN2, LRG1, S100P, and TACSTD2) that played key roles in the network, and we analyzed the expression of these nodes S100P in the GEPIA database. Consistent with the 5-hmC levels in CRC patient plasma, our external validation results from the GEPIA and UALCAN databases showed that LCN2, LRG1, S100P, and TACSTD2 were highly expressed in CRC tissue compared with controls. The DNA promoter methylation levels of LCN2, LRG1, and S100P were lower in CRC tissue than in normal control tissue.ConclusionThe present findings suggest that abnormality in cell-free DNA hydroxylation in plasma may be associated with CRC. In addition, the 5-hmC levels of LCN2, LRG1, S100P, and TACSTD2 in circulating cfDNA may be used as potential noninvasive markers for CRC.

Project description:BackgroundLiquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release.MethodsWe measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release.ResultsHigher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding.ConclusionsMethylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.

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Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer.

Publications

Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer.

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