Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy...

Area of Science:

• Oncology
• Molecular Biology
• Genetics

Background:

• Liquid biopsy offers real-time tumor evolution characterization via circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA).
• Epigenetic silencing of tumor suppressor genes is crucial for cancer progression and metastasis.
• Previous research established the presence of epigenetic alterations in CTCs.

Purpose of the Study:

• To develop and validate a sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA).
• To simultaneously detect methylation status of key suppressor genes (CST6, SOX17, BRMS1) in liquid biopsy samples and primary tumors.
• To analyze epigenetic changes in CTCs and ctDNA from breast cancer patients.

Main Methods:

• Development and analytical validation of the MMSPA technique.
• Simultaneous detection of methylation in CST6, SOX17, and BRMS1 genes.
• Analysis of EpCAM-positive CTCs, corresponding ctDNA, and paired primary breast tumors.

Main Results:

• Methylation of CST6, BRMS1, and SOX17 was observed in CTCs fractions of early breast cancer and metastatic patients.
• Similar methylation patterns were detected in the corresponding ctDNA.
• Specific methylation frequencies were quantified for each gene in both CTCs and ctDNA across patient groups.

Conclusions:

• Epigenetic alterations are prevalent in EpCAM-positive CTCs and ctDNA in breast cancer patients.
• The developed MMSPA methodology demonstrates high sensitivity and specificity.
• This approach holds potential for broader application in various cancer types and for detecting multiple gene targets.