title: Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy analysis.
abstract: BACKGROUND
  Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs.
  
  
  METHODS
  We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors.
  
  
  RESULTS
  In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively.
  
  
  CONCLUSIONS
  Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer.
authors: Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771, Greece.
authors: C
authors: C
authors: Parisi
authors: Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771, Greece.
authors: S
authors: S
authors: Mastoraki
authors: Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771, Greece.
authors: A
authors: A
authors: Markou
authors: Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771, Greece.
authors: A
authors: A
authors: Strati
authors: Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771, Greece.
authors: M
authors: M
authors: Chimonidou
authors: Department of Medical Oncology, University of Crete, 71110, Heraklion, Crete, Greece.
authors: V
authors: V
authors: Georgoulias
authors: Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771, Greece. Electronic address: lianidou@chem.uoa.gr.
authors: E S
authors: ES
authors: Lianidou
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics*; D001706:Biopsy; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics*; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects* / Q000235:genetics; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics*; D005260:Female; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm
keywords: Breast cancer; Circulating tumor DNA; Circulating tumor cells; DNA methylation; Liquid biopsy; Methylation specific PCR