43P - Assessment of methylation-specific genetic markers for reliable colorectal cancer detection and their potential in liquid biopsy applications

Background

DNA methylation alterations are extensively studied biomarkers in cancer diagnostics. CpG islands, DNA regions rich in methylable CG dinucleotides, predominantly occur in gene promoter regions. Their localization to well-defined regulatory DNA sequences makes them ideal candidates for investigating (epi)genetic aberrations using targeted detection techniques like droplet digital PCR (ddPCR). This pilot study aims to select and validate a panel of methylation markers frequently occurring in colorectal cancer (CRC), where methylation changes precede genetic mutations.

Methods

We focus on three biomarkers: branched-chain amino acid transaminase 1 (BCAT1), IKAROS family zinc finger 1 (IKZF1), and Septin 9 (SEPT9). Methylation status of BCAT1 and IKZF1 will be analyzed by examining CpG islands in promoter sequences of their longest protein-coding isoforms, while SEPT9 methylation will be examined in regulatory regions of alternatively spliced variants. The detection method employs methylation-specific real-time PCR (MethyLight) using fluorescent TaqMan probes. Amplification of predominant non-methylated DNA is suppressed using methylation-specific blocking oligonucleotides.

Results

Primer specificity was evaluated using gradient qPCR on tumor and adjacent non-tumor tissues. Markers were assessed on samples harboring common CRC mutations (KRAS, TP53, BRAF, APC), demonstrating 83% positivity for all three biomarkers. Efficient blocking of non-methylated DNA resulted in no detectable signal in non-tumor tissues, suggesting high primer specificity for tumor-specific methylation patterns.

Conclusions

Preliminary results show specific amplification of methylated regions exclusively in tumor tissue. The next phase will evaluate sensitivity and specificity of these markers in plasma samples from CRC patients using ddPCR technology. This approach aims to validate the potential utility of these methylation markers in liquid biopsy applications for CRC detection and monitoring.

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

The authors.

Funding

Ministry of Health, Czech Republic (MH CZ - DRO - VFN00064165).

Disclosure

All authors have declared no conflicts of interest.