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Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
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Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy

C Parisi1, S Mastoraki1, A Markou1

  • 1Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771, Greece.

Clinica Chimica Acta; International Journal of Clinical Chemistry
|August 12, 2016
PubMed
Summary

This study developed a novel liquid biopsy method to detect epigenetic alterations in breast cancer. The Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) shows promise for real-time tumor evolution monitoring.

Keywords:
Breast cancerCirculating tumor DNACirculating tumor cellsDNA methylationLiquid biopsyMethylation specific PCR

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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.