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

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.

Blood Free-Circulating DNA Testing of Methylated RUNX3 Is Useful for Diagnosing Early Gastric Cancer

“…The evaluation of the methylation level is performed with droplet digital PCR, which can count even one copy of the target gene [19]. These factors result in an experimental technique that is easier to perform in the CORD assay than in the conventional methylation assays [12][13][14][20][21][22]. The problem of the restriction enzyme-based methylation assay, including the CORD assay, is the possibility of incomplete digestion leading to false positives.…”

“…The use of the CORD assay is especially advantageous when using biobank resources to perform retrospective studies because the amounts in commercially available blood samples from biobanks are small, usually less than 1 mL [30]. Up to 4 mL of plasma or serum can be required for the conventional methylation assays of laboratory testing [21,22,31], whereas for a single test, the CORD assay only requires a DNA amount equivalent to that in 40 µL of serum. Thus, the measurement of the methylation levels of various genes in archived blood samples from a biobank and confirmation of the reproducibility of data are much easier with the CORD assay.…”

TERT structural rearrangements in metastatic pheochromocytomas

“…Furthermore, additional analysis is required to verify the canonical and non-canonical functions of TERT in PC/PGL tumors (Martinez & Blasco 2011). Finally, a number of therapeutic strategies (such as telomerase inhibition, TERT promoter-driven therapy and TERT-directed immunotherapy) are currently under investigation, which may lead to improved outcomes in individuals with TERT-overexpressing metastatic PC/PGL tumors (Xu & Goldkorn 2016).…”

Clinical Application of Circulating Tumor DNA in the Genetic Analysis of Patients with Advanced GIST

“…Compared with traditional tissue biopsy, liquid biopsy of ctDNA is noninvasive, simpler, and safer. The ctDNA level is associated with tumor burden, indicating a potential role of ctDNA as an informative, inherently specific, and highly sensitive biomarker in the diagnosis and prognosis of patients with various types of cancers (5)(6)(7). ctDNA detection has also been used to determine genotype in patients with GIST, which is beneficial for guiding targeted therapy (8).…”