Methylation profiles of 22 candidate genes in breast cancer using high-throughput MALDI-TOF mass array.

Alterations of DNA methylation patterns have been suggested as biomarkers for diagnostics and therapy of cancers. Every novel discovery in the epigenetic landscape and every development of an improved approach for accurate analysis of the events may offer new opportunity for the management of patients. Using a novel high-throughput mass spectrometry on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) silico-chips, we determined semiquantitative methylation changes of 22 candidate genes in breast cancer tissues. For the first time we analysed the methylation status of a total of 42 528 CpG dinucleotides on 22 genes in 96 different paraffin-embedded tissues (48 breast cancerous tissues and 48 paired normal tissues). A two-way hierarchical cluster analysis was used to classify methylation profiles. In this study, 10 hypermethylated genes (APC, BIN1, BMP6, BRCA1, CST6, ESRb, GSTP1, P16, P21 and TIMP3) were identified to distinguish between cancerous and normal tissues according to the extent of methylation. Individual assessment of the methylation status for each CpG dinucleotide indicated that cytosine hypermethylation in the cancerous tissue samples was mostly located near the consensus sequences of the transcription factor binding sites. These hypermethylated genes may serve as biomarkers for clinical molecular diagnosis and targeted treatments of patients with breast cancer.

Parallel assessment of circulatory cell-free DNA by PCR and nucleosomes by ELISA in breast tumors.

OBJECTIVES: In order to assess the potential biomolecules for breast cancer, we analyzed in parallel the levels of cell-free glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cell-free nucleosomes in serum samples from patients with benign and malignant breast tumors. The levels of cell-free DNA obtained by quantitative PCR were compared with those obtained by enzyme-linked immunosorbent assay (ELISA). METHODS: Twenty-three patients with benign breast tumors, 27 patients with breast cancer, and 32 age-matched healthy women were recruited. The amounts of serum nucleosomes were analyzed by ELISA and the levels of cell-free GAPDH were measured by real-time quantitative PCR. The correlation between nucleosome and cell-free GAPDH levels was examined using the Spearman rank test. RESULTS: The levels of cell-free GAPDH were significantly higher in the serum samples of patients with benign and malignant breast tumors than in those of the control group (median 37,966 GE/mL, range 3,802-130,104 versus 11,770 GE/mL, range 2,198-73,522, p=0.035 and median 40,698 GE/mL, range 3,644-192,482 versus 11,770 GE/mL range 2,198-73,522, p=0.001). The concentration of cell-free GAPDH correlated significantly with the quantities of nucleosomes in serum samples (r=0.451, p=0.000). There was, however, no significant difference between healthy individuals and women with benign breast tumors or breast cancer in terms of nucleosomes determined by ELISA. CONCLUSION: Our data suggest that the cell-free serum GAPDH DNA assayed by quantitative PCR is a better biomarker than nucleosomes assayed by ELISA in patients with breast tumors.