Breast Cancer Metastasis Suppressor-1 Promoter Methylation in Primary Breast Tumors and Corresponding Circulating Tumor Cells

Abstract

Breast Cancer Metastasis Suppressor-1 differentially regulates expression of multiple genes, leading to metastasis suppression without affecting orthotopic tumor growth. We evaluated BRMS1 promoter methylation as prognostic biomarker in primary breast tumors and studied BRMS1 promoter methylation in a subset of corresponding Circulating Tumor Cells (CTC) for the first time. We analyzed 118 formalin-fixed paraffin embedded samples: 5 pairs of breast tumors and adjacent non-cancerous tissues, 14 non-cancerous tissues, 10 benign fibroadenomas, and 84 primary breast tumors. Peripheral blood mononuclear cells from 39/84 of these patients were fixed in cytospins. BRMS1 methylation status was investigated in all FFPE and cytospin stained CTC using methylation specific PCR. BRMS1 expression in cytospins was examined by double-immunofluorescence using anti-BRMS1 and pancytokeratin A45-B/B3 antibodies. BRMS1 promoter methylation was not observed in noncancerous breast tissues (0%), and benign fibroadenomas (0%), while it was observed in 36.9% of primary breast tumors. BRMS1 promoter methylation in primary tumors was associated with reduced disease-free interval (P=0.009) while a trend towards a reduced overall survival was also observed (P=0.071). 13/39 cytospin samples (33.3%) were positive for the presence of CTC and the total number of the detected CTC was 41. Most CTC (80.5%) were negative for BRMS1 or maintained low expression, implying that BRMS1 is down regulated in these cells. BRMS1 promoter methylation was observed in 5/39 (12.8%) samples. BRMS1 promoter methylation in primary breast tumors provides prognostic information for DFS. BRMS1 expression in CTC was highly heterogeneous, between patients and even in the same patient.

Affinity Versus Label‐Free Isolation of Circulating Tumor Cells: Who Wins?

“…Chimonidou et al analyzed CTCs and cell‐free DNA in breast cancer for methylation of a tumor suppressor gene SOX17 promoter . In another study of breast cancer, methylation of a metastasis suppressor gene was studied in primary tumor and CTCs, and the authors investigated its effect on survival . Whilst the rarity of CTCs offers challenges in enrichment and downstream assay feasibilities, cell free DNA suffer from similar limitations with respect to available amounts .…”

Gene expression profiling and DNA methylation analyses of CTCs

“…In CTCs of patients with operable breast cancer, promoter methylation of BRMS1 was observed in 32.1%, while in CTCs of patients with verified metastasis, promoter methylation of BRMS1 was observed in 44.4% (Obermayr et al., 2013). BRMS1 promoter was found to be highly methylated in CTC, while immunofluorescence studies in the same cytospins have shown that it was down regulated at the protein level in CTC as well (Chimonidou et al., 2013b). Moreover, we have recently shown that methylation of BRMS1 promoter in circulating tumor DNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumor biomarker (Balgkouranidou et al., 2014).…”

Breast cancer metastasis suppressor-1 promoter methylation in cell-free DNA provides prognostic information in non-small cell lung cancer

“…The specificity and sensitivity of the MSP assay for BRMS1 promoter methylation has been previously verified (Chimonidou et al , 2011, 2013). …”

“…The methylation status of BRMS1 gene in tissue samples was detected by conventional MSP by using specific primer pairs for both the methylated and unmethylated promoter sequences as previously described (Chimonidou et al , 2013). Each MSP reaction was performed in a total volume of 25  μ l. In all, 1  μ l of SB-converted DNA was added into a 24  μ l reaction mixture that contained 0.1  μ l of Taq DNA polymerase (5 U  μ l −1 , hot start GoTaq Polymerase; Promega, Madison, WI, USA), 5  μ l of the supplied 10 × PCR buffer, 2.0  μ l of MgCl 2 (50 mmol l −1 ), 0.5  μ l of dNTP's (10 mmol l −1 ; Fermentas, Carlsbad, CA, USA) and 1  μ l of the corresponding forward and reverse primers (10  μ mol l −1 ); dH 2 O was added to a final volume of 25  μ l. Sodium bisulfite-treated DNA was amplified in two separate MSP reactions, one with a set of primers specific for the methylated and one for the unmethylated BRMS1 promoter sequences.…”