Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease

Abstract

Background Tumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis. Materials and methods We prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF. Results We found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/ neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001). Conclusion The detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.

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

“…Circulating tumour cells as a source of cancer-related DNA methylation were reported by Matuschek and colleagues. They observed a correlation between DNA methylation in APC and GSTP1 genes in serum with the presence of circulating blood tumour cells, which are associated with a more aggressive and advanced BC disease [49]. Our current results are in concordance with our previous study where we found different spectra of methylated genes in plasma compared to tumour samples, using the RT-based quantitative method [28].…”

Strategies for discovery and validation of methylated and hydroxymethylated DNA biomarkers

“…MethyLight is a high‐throughput, specific, sensitive, and quantitative assay that requires very small amounts of DNA; thus, it is suitable to be used in clinical laboratories. The utility of MethyLight for DNA methylation‐based biomarker has been demonstrated by numerous studies, including the methylation of GSTP 1, APC , TGFβ 2, HOXD 3, MLH 1, dickkopf homolog 1( DKK 1), and secreted frizzled‐related protein 1 ( SFRP 1), which has been shown to be detected in prostate and colon cancers . More recently, MethyLight has been improved with the implementation of digital PCR .…”

APC gene promoter aberrant methylation in serum as a biomarker for breast cancer diagnosis: A meta‐analysis

“…23,24 Recently, studies have also found that aberrant methylation of cancer-related genes can be detected in the peripheral blood or serum in patients with malignant carcinomas. 15,16 However, in healthy or non-cancerous subjects, aberrant methylation is rarely detected in the serum or blood. This indicates that the detection of aberrant serum methylation may represent a potential biomarker for BC diagnosis or screening.…”

“…Twelve publications relevant to APC gene promoter methylation and BC were identified and included in this study. 5,6,[8][9][10][11][12][13][14][15][16][17] The search process is shown in Figure 1. The ethnicity of the patients in the 12 studies was Caucasian (8), East Asian (3), and African (1).…”