Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay

Abstract

Aberrant promoter methylation of genes is a common molecular event in breast cancer. Thus, DNA methylation analysis is expected to be a new tool for cancer diagnosis. In this article, we have established a new, high-performance DNA methylation assay, the one-step methylation-specific polymerase chain reaction (OS-MSP) assay, which is optimized for analyzing gene methylation in serum DNA. The OS-MSP assay is designed to detect aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 genes in serum DNA. Moreover, two quality control markers were designed for monitoring the bisulfite conversion efficiency and measuring the DNA content in the serum. Serum samples were collected from patients with primary (n = 101, stages I-III) and metastatic breast cancers (n = 58) as well as from healthy controls (n = 87). If methylation of at least one of the three genes was observed, the OS-MSP assay was considered positive. The sensitivity of this assay was significantly higher than that of the assay involving conventional tumor markers (CEA and/or CA15-3) for stages I (24 vs. 8%) and II (26 vs. 8%) breast cancer and similar to that of the assay involving the conventional tumor markers for stage III (18 vs. 19%) and metastatic breast cancers (55 vs. 59%). The results of the OS-MSP assay and those of the assay involving CEA and/or CA15-3 seemed to compensate for each other because sensitivity of these assays increased to 78% when used in combination for metastatic breast cancer. In conclusion, we have developed a new OS-MSP assay with improved sensitivity and convenience; thus, this assay is more suitable for detecting aberrant promoter methylation in serum DNA. Moreover, the combination of the OS-MSP assay and the assay involving CEA and/or CA15-3 is promising for enhancing the sensitivity of diagnosis of metastatic breast cancer.

Methylated DNA and Total DNA in Serum Detected by One-Step Methylation-Specific PCR Is Predictive of Poor Prognosis for Breast Cancer Patients

“…The OS-MSP assay was conducted as previously described [20]. In brief, a total of 1,000 µl of serum from each patient was solubilized by incubation in lysis buffer (5 m guanidine-HCl and 0.65 mg/ml proteinase K) at 50°C for 1 h and then incubated with a 5 m bisulfite solution at 80°C for 40 min.…”

“…Quantification of total DNA was done as previously described [20]. In brief, in order to quantify total DNA in serum after bisulfite treatment, a genomic locus lacking a cytosine base was selected since such a locus is not affected by bisulfite treatment, and this locus was amplified by PCR using primers, probe, and standard oligo DNA.…”

“…One microgram of DNA was subjected to sodium bisulfite treatment using the EpiTect Bisulfite Kit (48; Qiagen) according to the manufacturer’s protocol and was analyzed by MSP for promoter hypermethylation of GSTP1 , RASSF1A , and RAR β 2 as previously described [20]. …”

“…Very recently, we have been able to develop the one-step MSP (OS-MSP) assay which is a more sensitive method for detection of met-DNA in serum of breast cancer patients [20]. In the OS-MSP assay, the first DNA extraction step from the serum is eliminated and the incubation time with bisulfite is shortened, while the methylated genes (GSTP1 , RASSF1A , and RAR β 2) can be detected with a sensitivity about 200 times higher than that of the cMSP assay.…”

Recent Progress in Triple Negative Breast Cancer Research

“…DNA hypermethylation studies in breast carcinoma have focused on the methylation status of some tumorrelated genes in invasive breast cancer as compared to normal breast tissue (Gheibi et al, 2012;Sturgeon et al, 2012;Yamamoto et al, 2012). Several studies have highlighted the epigenetic regulation of some genes including DAPK (gene associated with DNA apoptosis), TWIST, PAX5 and ID4 (transcription factors), GSTP1 (gene involved in detoxification pathway of xenobiotic), p16 (tumor suppressor gene), CDH13 (involved in cell adhesion) and RARβ (retinoic acid receptor).…”