Quantitative detection of methylated ESR1 and 14-3-3-σ gene promoters in serum as candidate biomarkers for diagnosis of breast cancer and evaluation of treatment efficacy

Abstract

The aim of the present study was to investigate the association between gene hypermethylation and main clinicopathological features of breast cancer, including diagnosis and treatment response. A sensitive SYBR green methylation-specific PCR technique was used to analyze the utility of circulating DNA with CpG island hypermethylation of ESR1, APC, RARB, 14-3-3-sigma and E-cad gene promoter regions as breast cancer biomarkers. Analyses were conducted of preoperative sera from 106 women with breast cancer, 34 with benign breast disease and 74 with no evidence of breast disease and of post-treatment sera from 60 of the breast cancer patients. Mean serum values of methylated ESR1 and 14-3-3-sigma gene promoters significantly differed between breast cancer patients and healthy controls (p = 0.0112 for ESR1 and p = 0.0047 for 14-3-3-sigma). When their results were combined, it was found that hypermethylation of these two genes differentiated between breast cancer patients and healthy controls (p < 0.0001) with a sensitivity of 81% (95% confidence interval: 72-88%) and specificity of 88% (95% CI: 78-94%). Presence of methylated ESR1 in serum of breast cancer patients was associated with the ER negative phenotype (p = 0.0179). Serum hypermethylation at ESR1 and 14-3-3-sigma loci was observed in cancer patients, in situ carcinoma and benign breast disease. No significant differences in methylated ERS1 or 14-3-3-sigma values were observed between pre-surgery and post-treatment measurements. Preliminary clinical applications of this approach have revealed several shortcomings, including a frequent presence of methylated 14-3-3-sigma in sera from women with breast benign disease. These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA remains a promising tool for cancer risk assessment.

Proteomic Changes Induced by Effective Chemopreventive Ratios of n-3:n-6 Fatty Acids and Tamoxifen against MNU-Induced Mammary Cancer in the Rat

“…3C). The protein 14-3-3 sigma, not identified by iTRAQ analysis but still of interest due to its well-known tumor suppressor activity in breast cancer (17)(18)(19), was observed to be increased significantly in the 10:1 and 25:1 n-3:n-6 groups (Fig. 3D).…”

“…This protein has been shown to be downregulated in many types of cancer including breast cancer (18,40). It is thought that hypermethylation at CpG islands at the gene promoter of the 14-3-3 sigma causes the downregulation of the protein in breast cancer patients (19). It was recently determined that docosahexaenoic acid increased the amount of 14-3-3 sigma protein expressed in colon cancer cells (41).…”

DNA methylation-based biomarkers in serum of patients with breast cancer

“…It is one of the most frequent and early methylated genes in the progression from normal to atypical hyperplasia, to DCIS and finally to invasive carcinoma. The utility of this tumor marker is partially compromised by contamination of methylated alleles in the stroma and peripheral white blood cells [115][116][117].…”

Circulating Tumor Nucleic Acids: Perspective in Breast Cancer

“…Importantly, AI or LOH were not observed in any of the healthy subjects whereas they were detectable in patients with carcinoma in situ [8] suggesting the potential application of this type of analysis in screening proce- had a much better outcome than the patients that reversed from negative to positive during treatment (an indicator of disease progression). This finding was recently confirmed utilizing two other genes: 14.3.3s and ESR1 [38]. Although the analysis of the methylation status of informative target sequences to monitor the efficacy of the therapy is still in its developmental stage, it is reasonable to hypothesize that a panel of methylated targets might be utilized as surrogate marker for circulating breast cancer cells.…”

“…In this respect, an increased risk of breast cancer has been documented in patients with fibroadenoma, and the presence of tumor-associated lesions in benign tumors has been documented [8,36,37]. Interestingly, tumor-associated alterations were detected in the serum of patients with in situ carcinoma and with benign breast disease suggesting that this approach might have a supporting role in the early diagnosis of breast cancer [35,37,38].…”