Neoplasma. 2013;60(6):635-46. doi: 10.4149/neo_2013_082.
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.
乳腺癌是最常见的癌症之一，其死亡率高的原因是转移性疾病。因此，新的分子生物标志物预测肿瘤的转移潜能将有助于预防转移和个性化治疗。本研究的目的是探讨侵袭性和转移相关基因的 DNA 甲基化水平与异常蛋白表达之间的关系，以及是否在肿瘤和循环无细胞 DNA 中存在类似的 DNA 甲基化水平，从而利用血浆 DNA 甲基化作为预后生物标志物。我们使用焦磷酸测序法分析了 34 名原发性乳腺癌患者的肿瘤、血浆和外周血细胞中 11 个基因（APC、ADAM23、CXCL12、ESR1、PGR B、CDH1、RASSF1A、SYK、TIMP3、BRMS1 和 SOCS1）的 DNA 甲基化水平，以及 50 名健康对照者的血浆和外周血细胞。同时，通过免疫组织化学评估石蜡包埋肿瘤样本中相关蛋白的表达。统计分析采用 SPSS statistics 15.0 软件。结果发现，肿瘤中最常见的甲基化基因是 RASSF1A（71.9%）、APC（55.9%）、ADAM23（38%）和 CXCL12（34.4%），甲基化水平高达 86%、86%、53%和 64%。与患者外周血细胞核 DNA 相比，肿瘤中发现 9 个基因的甲基化水平显著升高。此外，在患者外周血细胞核 DNA 中，CXCL12、ESR1 和 TIMP3 基因的甲基化水平明显高于对照组，但未超过 15%。另一方面，患者肿瘤中 DNA 甲基化与无细胞血浆 DNA 之间未观察到相关性。此外，在患者和对照组中，血浆样本中累积 DNA 甲基化的数值（43.6%±20.1%对 43.7%±15.0%）几乎相同。在所有评估的蛋白中，肿瘤组织均呈现出从高表达到无表达的不同谱，但在 APC 和 CXCL12 基因中，观察到随着相应蛋白表达的增加，平均 DNA 甲基化水平呈下降趋势。我们研究小组的乳腺癌 DNA 甲基化谱是癌症特异性的，但它们不是唯一影响评估基因沉默和相关蛋白产物减少的原因。外周血细胞核 DNA 中 DNA 甲基化检测在癌症诊断和治疗中的临床应用尚需进一步研究。
