DNA methylation biomarkers for minimally invasive and early detection of colorectal cancer using liquid biopsies

Background: Colorectal cancer (CRC) is the 3rd most common cancer worldwide, and prognosis is highly dependent on the stage of the cancer at the time of diagnosis. Therefore, early diagnosis of colorectal cancer using minimally invasive sampling techniques is of great importance. In the presented work we followed an approach in which we developed a DNA methylation biomarker panel that can simplify and improve the early diagnosis of colorectal cancer using liquid biopsy. Detection of tumor specific DNA methylation marks in cfDNA from liquid biopsy has several advantages over other biomarkers and has great potential to solve diagnostic needs.
Methods: We setup a multiplexed µ-fluidic-MSRE-qPCR workflow on the Biomark-System (Standard Biotools) for DNA methylation analysis using cfDNA from liquid biopsies. As a first step, we tested the workflow with 48 DNA methylation markers in liquid biopsy samples from 88 patients (44 CRC, 44 controls). The established workflow was then used to perform 180plex analysis on 1000 liquid biopsy samples to select a robust qPCR-based DNA methylation marker panel for early diagnosis of CRC.
Results: Preliminary experiments showed that the cfDNA contained in 2ml plasma was sufficient to test up to 96 DNA methylation markers in parallel. ROC-Analysis of the 48 candidate markers on 44 patients with CRC and 44 control samples resulted in AUC>0.85 for WT1, PENK, SPARC, GDNF, TMEFF2, and DCC. Applying this workflow to 180 markers and 1000 liquid biopsy samples yielded a robust diagnostic DNA methylation 12-plex classifier providing overall CRC detection with a sensitivity of 0.77 (up to 100% depending on tumor stage) and a specificity of 0.88.
Conclusions: DNA methylation testing using liquid biopsy is a promising tool for the early detection of CRC and can be used in routine testing procedure as it is inexpensive and easy to use.