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P01 Molecular investigation of the DNA methylation status of one four-gene panel in greek patients from breast FNAC samples
  1. E Vavoulidis1,
  2. N Kougioumtsidou1,
  3. M Nasioutziki1,
  4. E Mareti1,
  5. GC Pratilas1,
  6. A Liberis1,
  7. S Petousis1,
  8. E Chasioti1,
  9. A Daniilidis1,
  10. A Papanikolaou1,
  11. L Zepiridis2 and
  12. K Dinas1
  1. 12nd Obstetrics and Gynaecology
  2. 21rst Obstetrics and Gynaecology, Aristotle University of Thessaloniki, Thessaloniki, Greece


Introduction/Background Alterations in DNA methylation pattern are common in various cancers including, breast tumors. Aberrant methylation of normally unmethylated CpG-rich areas which are in/near the promoter region, has been associated with transcriptional inactivation of various genes in breast cancer patients, with most studies focusing on tissue samples. In this study, we determined the frequency of aberrant promoter methylation of the APC, CDH13, HIN-1 & CYCLIND2 genes through molecular analysis of cytological material obtained with fine needle aspiration (FNAC).

Methodology Our study retrospectively analyzed 103 women who visited our Breast Pathology Clinic. FNAC was collected in Cytolyt (Hologic). DNA was extracted from breast cells with Quick-gDNA MiniPrep kit (Zymo) and was subjected to complete bisulfite conversion with EZ DNA Methylation-Gold Kit (Zymo). A methylation specific PCR was carried out with primers designed for amplification of either the methylated or the unmethylated condition for each gene. The final PCR-products were separated in 2% agarose gels with electrophoresis.

Results DNA methylation was observed for all 4 genes in over 75% of the C4 and C5 cytologically-classified samples (n=66). In the ‘gray zone’ of C3 cytologically-classified samples (n=11), the methylation frequencies were 72.7% for CND2 & APC and 63.7% for CDH13 & HIN1. In comparison, the vast majority of C1 and C2 cytologically-classified samples were unmethylated for all 4 genes with methylation frequencies ranging from 0% to 25%.

Conclusion It seems that in patients with C3 to C5 cytological diagnosis, high methylation frequencies were observed for all candidate genes in contrast with the low frequencies in patients negative for neoplasia or with benign breast lesions. A multiplex genetic panel for detection of DNA methylation, combined with cytological diagnosis and other clinical/lifestyle parameters, during a non-operative FNA could provide important information for clinicians pre-operatively leading to a more accurate detection and staging of breast tumors.

Disclosure Nothing to disclose.

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