Hypermethylation at CpG islands of GSTP1 gene’s promoter is the typical property of breast cancer in Vietnamese population

The demand for biomarker that applied in prognosis, early diagnosis, predicting and/or monitoring the therapeutic response and detection of recurrent cancer is the worldwide expection. One of the biomarker, which the worldwide researches focused on, is the hypermethylation at CpG island of promoter which undergoes the DNA methylation changes in carcinogenesis. In current study, the target gene is GSTP1 (Glutathion-Stransferase pi 1 gene), with one of the most function is particularly involved in detoxification. Because of this function, GSTP1 (obviously based on the frequencies of methylation) has been chosen as a potential candidate gene for several clinical trials in predicting of the response to the demethylation drugs in several cancers, including breast cancer. In this study, we foscused on the evaluation of the methylation status of 115 biopsy specimens collected from university medical center HCMC, including 95 breast cancer specimens and 20 noncancerous breast tissue. The results showed that 41 of 95 (43.2 %) breast cancer specimens are hypermethylated, no methylation was found on the noncancerous breast tissue (p<0.01). These results allowed us to predict the totally corresponding to the application of the treatment, which based on the demethylation drug, one of the epi-drug line, in Vietnamese patients in the near future.

Hypermethylation at CpG islands of GSTP1 gene's promoter is the typical property of breast cancer in Vietnamese population

ASTRACT
The demand for biomarker that applied in prognosis, early diagnosis, predicting and/or monitoring the therapeutic response and detection of recurrent cancer is the worldwide expection. One of the biomarker, which the worldwide researches focused on, is the hypermethylation at CpG island of promoter which undergoes the DNA methylation changes in carcinogenesis. In current study, the target gene is GSTP1 (Glutathion-Stransferase pi 1 gene), with one of the most function is particularly involved in detoxification. Because

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Breast cancer is the most frequent cancer among women in Vietnam. According to the Globocan (2012), it was observed that, in 2012, people newly diagnosed with breast cancer has significant increased to 125,000. Currently, although chemotherapy or chemoradiotherapy are the most effective treatments of breast cancer, due to the patient's clinical characteristics, genetic differences in drug transported, therefore it is limited by significant heteogeneity variation in response and toxicity [6,7,21]. For further innovatory therapy, an understanding of the molecular events as an aberrant epigenetic modification, leading to the development of cancer, including breast cancer, is the worldwide expectation. DNA hypermethylation has been reported as the potential biomarker for the diagnosis and prognosis of cancer [1,10], it was involved in the GSTP1 case. The hypermethylation of GSTP1 promoter was also detected in several cancers including breast cancer, prostate cancer, lung cancer… The frequency of the hypermethylation of GSTP1 was found out as 26 % [17], 18 % [15] in breast cancer and 36.1 % [11] in prostate cancer… Nowadays, the DNA methylation biomarker is also considered as an application in predicting and/or monitoring the therapeutic response, as well as detection of recurrent cancer, because DNA methylation changes were observed during the carcinogenesis [10].
GSTP1 (Glutathion S-transferase P1), which is belonged to the tumor suppressor genes family, involves in the detoxification of reactive carcinogen metabolites and plays an important role in the cellular defense system [2,17,21]. The hypermethylation of GSTP1 has been reported as the potential DNA-based biomarkers in cancer, including breast cancer, and applied in predicting the epi-drug response [10]. Several studies showed that the hypermethylation of the CpG islands belonged to the promoter region of GSTP1 leading to the decrease in gene expression, resulted in breast cancer [2,3,5,17]. For the aims to improve the further successful promising biomarkers in Vietnamese population, in present study, we determined quantitatively the methylated frequency of GSTP1 gene's promoter and evaluated the correlation with the clinicopathological data of Vietnamese breast cancer patients.

Sample collection
A total of 115 samples including 95 breast cancer specimens and 20 non-cancer samples were collected and admitted from Ho Chi Minh city Medical hosptital center. All the breast cancer specimens were reviewed and immunohistochemical analysed with two predictive factors including HER2/neu and p53 antibodies (Ventana CONFIRM anti-HER-2/neu (4B5) and Bp53-11) according to the protocols of ASCO quality guideline [13]. 20 healthy specimens were obtained from women who were underwent the biopsy of the mammary gland because of mammographic screening and for whom histology confirmed the presence of only normal tissue. These tissues were obtained from the surgical progress, and then, embedded in the paraffin and finally, stored at -20 o C for further usage.

DNA extraction, bisufite modification and MSP assay
Total of genomic DNA was extraeted with phenol/chloroform method from all the paraffin embedded breast cancer specimens and the healthy samples. The concentration and purity of DNA extraction were analysed by the absorbance at 260 nm and 280 nm. The purity of which sample with the ratio OD260/OD280 values of 1.8 to 2.0 was used to the bisulfite modification by using the DNA modification kit (Epitect Kit, Qiagen).
For MSP (methylation specific PCR) assay, the primer set for MSP analysis of GSTP1 was used to qualify the methylation status of methylation and unmethylation of given gene (

Statistical analysis
The methylation frequency of GSTP1 was calculated and the differences in the presence of methylation were determined by two sided Fisher test and Chi squared tests for variables. Moreover, the OR (Odd ratio) and 95 %CIs (confidence intervals) were also calculated. Statistical analyses were performed by using

RESULTS AND DISCUSSIONS
The methylation frequency was qualified in total of 95 breast cancer specimens and 20 noncancer specimens. As the result, the frequency of methylated and unmethylated GSTP1 was 43.2 % (41 cases of 95 specimens). Otherwise, in noncancer specimens, none of methylation status was found (Fig. 1).  The methylation status was confirmed by DNA sequencing (Fig. 2). By DNA sequencing, we totally observed 9 CpG sites, which were totally methylated, were in examined region including regions 2 and 3. Specially, we detected 4 methylated CpG sites in the region 1 which was according to the binding site of the methylated reverse primer. Concerning to the signal of peaks in MSP product sequencing, the signals were unique, clear and specific. Based on these results, it was also concluded that the bisulfite modification was successful carried out.
In the context of methylation status, the aberrant methylation status of GSTP1 has been reported in many cancer including breast cancer, comparison to the recent report, the methylation frequency of GSTP1 in the present study was higher than the research of Yoon et al. (2012) [20] as 27.8 %, Lasabova et al. (2010) [14] as 24.45 %. Especially, comparison to a research in Asian country such as Thailand by Pongtheerat et al. (2011) [17], the methylation status of GSTP1 was also higher than Ponthreeat's research as 26 %. To evaluate the correlation between the DNA methylation and clinical parameters, the immunochemical staining of HER2/neu, p53 were carried out (Table 2). Because the overexpression of HER2/neu was found to be present in 15 % -30 % newly diagnosed breast cancer and mutations of p53 gene and/or p53 has been observed in 20 % -50 % of primary breast carcinomas [9,19], thus the expression of HER2/neu and p53 were used as the input values to confirm the breast cancer status Regarding to the DNA hypermethylation and HER/neu staining, we observed that there was not associated with the HER2/neu (p = 0.06). By contrast, the overexpression of p53 was strongly correlated with the methylation status (p = 0.0003). Interesting, taken these two prognosis biomarkers together, the frequent methylation of GSTP1 promoter hypermethylation in breast cancer were strongly associated to HER2/neu(-)p53(-) (p = 0.03). It meant that the DNA based marker methylation was negative associated with the other protein based markers.
In addition, to evaluate the applicability of GSTP1 methylation in breast cancer patients as biomarkers for breast cancer, the odd ratio (OR) was calculated. Even though only 20 non-cancer specimens were compared with 95 tumor specimens, we tentatively applied the Chi 2 test to calculate the OR value. As the result, we found out OR was 31.22 at 95% confidence with the significant statistic (p = 0.02). Moreover, the significant correlation between OR and breast cancer was considered. It meant that in the model the odds for a positive hypermethylation of GSTP1 in breast cancer were 31.22 times higher than in the cases of cancer without GSTP1 methylated. Thus, due to those results, it was noted that the hypermethylation of GSTP1 promoter was the specific characteristic of Vietnamese breast cancer patients. It is highlighted that this characteristic was adapted to not only as the potential biomarker in prognosis, diagnosis but also predicting/monitoring the demethylation drug responses as the function of GSTP1, in Vietnamese patients in the near future.