Evaluation of protein expression and DNA methylation profiles detected by pyrosequencing in invasive breast cancer

Abstract

Breast carcinoma is the most common cancer with high mortality caused by metastatic disease. New molecular biomarkers predicting the tumour's metastatic potential would therefore improve metastasis prevention and personalised care. The aim of the study was to investigate the relationship between DNA methylation levels in invasivity and metastasising associated genes with aberrant protein expression and also to evaluate whether a similar DNA methylation level is present in the tumour and circulating cell-free DNA for utilising plasma DNA methylation as prognostic biomarker. By using pyrosequencing, we analysed DNA methylation levels of 11 genes, namely APC, ADAM23, CXCL12, ESR1, PGR B, CDH1, RASSF1A, SYK, TIMP3, BRMS1 and SOCS1 in tumour, plasma and peripheral blood cells from 34 patients with primary breast cancer, as well as plasma and peripheral blood cells from 50 healthy controls. Simultaneously, the expression of related proteins in paraffin-embedded tumour samples was evaluated by immunohistochemistry. Statistical analysis was performed by SPSS statistics 15.0 software. Tumour DNA hypermethylation was found in most commonly methylated RASSF1A (71.9%), APC (55.9%), ADAM23 (38%) and CXCL12 (34.4%) genes with methylation levels up to 86, 86, 53 and 64 %, respectively. In tumours, significantly higher methylation levels were found in nine genes, compared with the patients´ peripheral blood cell DNA. Furthermore, in patients methylation levels in peripheral blood cell DNA were significantly higher than in controls in CXCL12, ESR1 and TIMP3 genes, but the values did not exceed 15%. On the other hand, no correlations were observed in patients between DNA methylation in tumours and cell-free plasma DNA. Moreover, in patients and controls nearly identical values of cumulative DNA methylation (43.6 % ± 20.1 vs. 43.7 % ± 15.0) were observed in plasma samples. A variable spectrum from high to none expressions presented in tumour tissues in all of the proteins evaluated, however in APC and CXCL12 genes a visible decreasing trend of mean DNA methylation level with increasing expression of the corresponding protein was observed. The DNA methylation profiles manifested in our group of breast carcinomas are cancer specific, but they are not the only cause that affects the silencing of evaluated genes and the decrease of relevant protein products. The clinical utility of DNA methylation testing in peripheral blood cell DNA for cancer diagnosis and therapy need to be further investigated.

CXCL12 and ADAM23 hypermethylation are associated with advanced breast cancers

“…In our group of patients, RASSF1A and APC hypermethylation was identified in 75.5% and 69.9% of the patients, respectively. In RASSF1A gene, we previously found the frequency from 71.9% to 82.6% 35,39,40 and similar results were observed in other studies, with the frequencies of RASSF1A methylation at 82.5% and 68% and of APC at 52.5%. 41,42 When compared with our results, the frequencies of the methylated BC samples from other studies were different for the CXCL12 gene (25.2% vs 14.5% and 52.4%, respectively), 23,24 and for ADAM23 (50.4% vs 69.2%), 22 but these authors only used qualitative methods for their methylation analyses.…”

“…The primary antibody specification and the laboratory procedures used were previously described. 35 The results are expressed as an immunoreactive score (IRS), also known as the German IRS, which evaluates both the percentage of positive cells and the staining intensity of the nuclei or the cytoplasm. 38 The staining intensity and percentage of positive cells were multiplied, giving 4 IRS categories: negative, weak, moderate, and high protein expression.…”

“…The primer sequences and the polymerase chain reaction conditions used were previously reported. 35 The pyrosequencing analyses were performed using a PyroMark Q24 System (Qiagen), and the results were evaluated using the PyroMark Q24 2.0.6. software (Qiagen). The methylation data are presented as the percentage of the average methylation in all the CpG sites in each gene.…”

“…The PL from the blood samples was processed and stored as previously described. 35 The cfDNA from the PL was isolated using a QIAamp DSP Virus Kit (Qiagen, Hilden, Germany), and the PBC DNA was obtained using a FlexiGene DNA Kit (Qiagen). For the DNA isolation from the paraffin-embedded PTs (.80% cancer cells), LNMs, and healthy breast tissue samples, the representative samples were selected by a pathologist, 3-mm-diameter cores from the blocked region were cut and the DNA was isolated using the MagneSil Genomic, Fixed Tissue System (Promega, Madison, Wisconsin).…”

Effect of genetics, epigenetics and variations in the transcriptional expression of cadherin–E in breast cancer susceptibility

“…Contrario a lo aquí reportado, en los estudios de Sturgeon, et al, y de Zmetakova, et al, no se asoció la metilación del promotor del CDH1 con el cáncer de mama (29,30). En cuanto a las características histopatológicas del tumor, se observó una asociación entre el estado de la metilación del promotor y los estados iniciales del tumor, caracterizados por ausencia de metástasis y un menor tamaño del tumor, lo cual evidenciaría la necesidad de la célula maligna de inactivar la cadherina E en las primeras fases del proceso de carcinogénesis.…”