Evaluation of Methylation Biomarkers for Detection of Circulating Tumor DNA and Application to Colorectal Cancer

Susan M Mitchell, Thu Ho, Glenn S Brown, Rohan T Baker, Melissa L Thomas, Aidan McEvoy, Zheng-Zhou Xu, Jason P Ross, Trevor J Lockett, Graeme P Young, Lawrence C LaPointe, Susanne K Pedersen, Peter L Molloy, Susan M Mitchell, Thu Ho, Glenn S Brown, Rohan T Baker, Melissa L Thomas, Aidan McEvoy, Zheng-Zhou Xu, Jason P Ross, Trevor J Lockett, Graeme P Young, Lawrence C LaPointe, Susanne K Pedersen, Peter L Molloy

Abstract

Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA) isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC), it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes) was analyzed in WBC DNA and eight differentially-methylated regions (DMRs) were taken through to testing in clinical samples using methylation specific PCR assays. DMRs residing in four genes, BCAT1, GRASP, IKZF1 and IRF4, exhibited low positivity, 3.5% to 7%, in the plasma of colonoscopy-confirmed healthy subjects, with the sensitivity for detection of ctDNA in colonoscopy-confirmed patients with colorectal cancer being 65%, 54.5%, 67.6% and 59% respectively.

Keywords: BCAT1; GRASP; IKZF1; IRF4 SDC2; SEPT9; biomarkers; circulating tumor DNA; colorectal cancer; epigenetics; hypermethylation; liquid biopsy; plasma.

Conflict of interest statement

R.T.B., M.L.T., A.M., L.C.P. and S.K.P. are current or past employees of and G.P.Y. is a consultant for Clinical Genomics Pty Ltd. The work described was planned and conducted jointly by employees of Clinical Genomics Pty Ltd. and of CSIRO.

Figures

Figure 1
Figure 1
Flowchart for evaluation of methylation in white blood cell (WBC) DNA. DNA was sheared and the methylated fraction was isolated using methylated DNA binding domain-capture (MBD-Cap). The captured DNA and the unbound fraction were separately bisulphite converted. Separate conversion-specific PCRs for each gene were performed to amplify both methylated and unmethylated sequences from each of the bound and unbound fractions.
Figure 2
Figure 2
Amplicon melt curve analysis. For each amplicon, melt curves of triplicate amplifications of pooled male and female DNA (six samples in all) from the DNA fraction captured by MBD-Cap (red traces) and DNA not captured (green traces) are shown. Arrows indicated the melt peak positions for control unmethylated (U) and fully methylated (M) DNAs.

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