Cpg Island Methylation of the MLH1, MGMT, DAPK, and CASP8 genes in Cancerous and adjacent noncancerous stomach tissues

Rita Kupčinskaitė-Noreikienė1, Jurgita Skiecevičienė2, laimas Jonaitis2, Rasa Ugenskienė1, Juozas Kupčinskas2, Rytis Markelis3, Vidmantas Baltrėnas4, Linas Šakavičius4, Irina semakina5, Saulius Grižas3, Elona Juozaitytė1 1Institute of Oncology, Medical Academy, Lithuanian University of Health Sciences, 2Institute for Digestive Research, Medical Academy, Lithuanian University of Health Sciences, 3Department of Surgery, Medical Academy, Hospital of Lithuanian University of Health Sciences, 4Department of Pathologic Anatomy, Medical Academy, Lithuanian University of Health Sciences, 5Department of Anaesthesiology, Medical Academy, Lithuanian University of Health Sciences, Lithuania


Introduction
Gastric cancer is the fourth most common malignancy and the third leading cause of cancer death in men and the fifth leading cause in women (1).
Many factors are involved in the development of gastric adenocarcinoma. Genetic host (2) and environmental factors, including Helicobacter pylori (H. pylori) infection (3), Epstein-Barr virus (4), diet (5), synergize and promote carcinogenesis pathways. However, the regulatory mechanisms involved in the development of gastric cancer remain poorly understood.
An epigenetic event -CpG island methylation of apoptosis and mismatch repair genes by the loss of their function -plays an important role in the development and progression of gastric adenocarci-noma. Epigenetic alterations also affect the expression of cancer genes alone or in combination with genetic mechanisms. The cytosine methylation of CpG dinucleotides in gene promoters is a common cause of DNA silencing and transcriptional repression that can modulate the clinical features of gastric cancer. Recent studies in the Asian population have indicated an important role of gene methylation in the cancer development and clinical variables.
Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase that participates in apoptosis pathways (6). DAPK methylation occurs more frequently in H. pylori-positive gastric cancer patients (7,8). There is an inverse correlation between DAPK methylation and microsatellite instability (MSI) (9). Gene promoter methylation alone or combined with other methylated genes serves as a predictive marker (lower response rate to fluoropyrimidine-based chemotherapy, shorter progression-free survival [PFS] in a metastatic setting), meanwhile DAPK methylation can be a prognostic marker related to shorter overall survival (10,11).
CASP8 is a member of the caspase family, which plays a central role in the execution phase of cell apoptosis. There is a lack of data about methylation frequency in the corresponding nontumor gastric tissue and associations with tumor histological characteristics and TNM grading.
The methylation of the DNA repair gene of O(6)methylguanine-DNA methyltransferase (MGMT) is important for cancer development. Gene promoter methylation is associated with cagA and vacA, which are virulence factors of H. pylori infection (12). MGMT promoter methylation in patients with gastric carcinoma was found to be associated with the mutations of the KRas gene, tumor stage, and disease-free survival (DFS) (13). Recent data show that MGMT promoter methylation is also a prognostic marker, and it is related to poor prognosis (14).
The importance of methylation of the mentioned genes in the pathogenesis of gastric cancer is significant. More data are becoming available regarding its prognostic and predictive value. Methylation frequency varies greatly, and most data come from East Asia. Therefore, it is important to determine the methylation frequency of gastric cancer gene promoters in Europe, to compare it with the methylation frequency of surrounding tissues, to determine a possible association with clinicopathological characteristics, and to determine the association between the methylation of individual genes.

Material and Methods
Study Population. Patients with histologically confirmed gastric adenocarcinoma were recruited at the Hospital of Lithuanian University of Health Sciences during the period of 2009-2011. Tissue samples were obtained by endoscopy or surgical resection from the tumor and the tumor-free area, which was at least 2 cm distant from the tumor and which was confirmed to be without any tumor cell infiltration by a histological assessment. Gastric tissue specimens were frozen in liquid nitrogen after dissection and stored at -80°C until analysis. Tumors were staged according to the criteria of the 2002 UICC/AJCC staging system for gastric cancer (21), and histologically subtyped and graded according to the World Health Organization (22) and the Lauren classification (23). Written informed consent was obtained from all study participants. The study was approved by Kaunas Regional Research Bioethical Methylation-Specific Polymerase Chain Reaction. DNA was extracted from 25-30 mg of frozen tissue using a ZS Genomic DNA™ Tissue Mini Prep Kit (Zymo Research, USA) according to the manufacturer's instructions. The methylation status of MLH1, MGMT, DAPK, and CASP8 gene promoters was determined by bisulfite treatment of DNA. Bisulfite treatment was performed using an EZ DNA Methylation Gold Kit™ (Zymo Research, USA) according to the manufacturer's instructions/ protocol. Human genomic DNA from peripheral blood lymphocytes treated with bisulfite served as a negative control. Human genomic DNA treated in vitro with Sss I methyltransferase (New England Biolabs, UK) was used as a positive control. The methylation status of the promoters was detected by methylation-specific polymerase chain reaction (MSP). The methylated and unmethylated DNA sequence primers are listed in Table 1. PCR was performed in a total volume of 20 μL, containing 10 μL Maxima® Hot Start PCR Master Mix (PCR buffer, dNTP, MgCl 2 ) with Hot Start Taq DNA polymerase (Thermo Fisher Scientific, USA), 10 μM of each primer (Metabion International AG, Germany), and 2 μL of converted DNA. MSP included 38-40 cycles starting at 94°C for 30 seconds, annealing at temperature appropriate for an individual gene (DAPK, 60°C; MLH1, 56°C; MGMT, 61°C; and CASP8, 58°C) for 1 minute, and extension at 72°C for 1 minute. The PCR products were separated by 3.5% gel electrophoresis. If both methylated and unmethylated signals appeared in a gel, the methylation of the gene was considered.
Statistical Analysis. Statistical analysis was carried out with the IBM SPSS Statistics 19 software (IBM SPSS Inc., Chicago, IL). Quantitative data are presented as mean and standard deviation (SD). For testing statistical hypothesis about the independence of two variables, the chi-square test or the Fisher exact test was used. A Spearman coefficient was calculated to determine correlation. The significance level of <0.05 was selected.

Results
The study population comprised 69 patients (39 men and 30 women) with a median age of 64.5 years (SD, 12.7; range, 23-87). The representative cases of methylation are shown in Fig. The methylation frequencies of MLH1, MGMT, DAPK, and CASP8 in the gastric cancer and paired nontumor tissues are presented in Table 2. The methylation of the CASP8 gene promoter was found to be quite a rare event both in the gastric cancer and the surrounding noncancerous tissue. There were no significant differences in the methylation frequency of the gene promoters between the can-cerous and adjacent noncancerous tissues.
An inverse correlation between the methylation of the DAPK and MLH1 gene promoters was observed in the cancerous (Spearman coefficient, -0.28; P=0.02) and surrounding noncancerous tissues (Spearman coefficient, -0.28; P=0.04). No significant associations between the methylation status of the studied genes and clinicopathological characteristics were found (Table 3).

Discussion
Our study and the study by Ivanauskas et al. (26) provide more information about the importance of gene CpG island methylation in gastric cancer in our region. The methylation frequency of DAPK in gastric cancer tissues varies from 22% to 91% (6-7, 10, 27-31). A study by Ye et al. reported that the methylation frequency of the DAPK promoter was significantly higher in the gastric cancer tissue than the corresponding nontumor tissue (30). The results of our study show a similar methylation frequency of the DAPK gene promoter in cancerous and adjacent noncancerous tissue. There are data available showing that the methylation of apoptosis-related genes correlates with poorly differentiated tumors and the advanced TNM stage (31), but we did not find any significant association between the methylation of the DAPK gene promoter and any pathological characteristics (TNM stage, Lauren tumor type, degree of differentiation G, involvement of lymph nodes) as well as clinical characteristics (age, sex). These findings, however, could be biased by a small sample size in our study, making the comparison between different subgroups difficult.
Only few articles about the importance of meth-  ylation of the CASP8 gene promoter in gastric cancer have been published. Our data showed that the methylation of the CASP8 gene promoter was quite a rare event in gastric cancer and surrounding noncancerous tissues. In both paired tissues, the methylation frequency was around 6%, and no significant difference between the compared tissues was observed. The methylation frequency of the apoptosis-related CASP8 gene promoter in this study was lower compared with that of the South Korean study that reported the methylation frequency of the CASP8 gene promoter in the gastric cancer and healthy tissues to be 16.7% and 4.2%, respectively (32). No significant associations between the methylation frequency of the CASP8 gene promoter and any pathological characteristics (TNM stage, Lauren tumor type, degree of differentiation G, involvement of lymph nodes) as well as demographic characteristics (age, sex) were found. The reported methylation frequency of the MGMT promoter in gastric cancer ranges from 6.9% to 61% (15,(27)(28)(33)(34), while it is reported to be 5.7% in the adjacent nontumor tissue (28). Our study showed a higher methylation frequency of MGMT in the nonmalignant (44.9%) than malignant (36.2%) tissues, but the difference was not significant. It could be related to technical aspects as our test was not quantitative but qualitative. Some data indicate that the methylation of the MGMT gene occurs more frequently in lymph node-positive gastric cancer (13,35). Our results do not contradict the literature data, but are not statistically significant. In line with the above mentioned genes, no significant association between the meth-ylation of the MGMT gene promoter and clinicopathological characteristics of patients with gastric cancer was found as well.
According to the published data, the methylation frequency of the MLH1 gene promoter in gastric cancer tissues varies greatly, i.e. from 14% to 65.3% (9,34,35). However, the methylation frequency of the MLH1 gene promoter determined in our study was higher compared to the previously published results in the European study by Balassiano et al. (31.9% and 14.2%, respectively) (34). Some data have indicated that the methylation of the MLH1 gene promoter occurs more frequently in the gastric cancer tissue compared with the adjacent noncancerous mucosa (35). In our study, the difference in the methylation frequency between cancerous and noncancerous tissues was 4%, but it was not significant. The methylation of MLH1 could be a diagnostic marker for gastric cancer, but further studies involving a larger number of patients are needed to confirm this, as the methylation frequency of MLH1 in cases of chronic gastritis is very low and does not reach 2% (24). A high methylation rate of surrounding noncancerous gastric tissues may indicate an association with local relapse frequency. This hypothesis should also be tested in future research. A Polish study reported that the MLH1 gene was hypermethylated more frequently in women than men (36), this was not confirmed in our study. Some authors detected the link between the methylation of the MLH1 gene promoter and the type of intestinal gastric cancer, lower clinical stage, absence of lymph node metastasis (9, 34, 35, 37,38). Contrary to these studies, our study failed to show the association between the methylation status of the mentioned gene and pathological characteristics of cancer such as tumor differentiation, tumor type by the Lauren classification, degree of differentiation G, and TNM staging. Data show that the methylation of the MLH1 gene promoter is associated with MSI (9,(15)(16)(17)(18)(19). Our results indicate that the methylation frequency of MLH1 inversely correlated with that of the DAPK gene promoter, which corresponds to the data presented in a study by Ferrasi et al. (9), reporting an inverse correlation between DAPK hypermethylation and MSI. This correlation was also confirmed in the surrounding noncancerous gastric tissue in our study.
Our study design has certain limitations. Further studies are needed to compare methylation patterns in gastric adenocarcinoma and adjacent tumor-free tissues with those of tissue specimens obtained from a healthy control group. The assessment of the gene methylation pattern in premalignant gastric lesions (atrophic gastritis and intestinal metaplasia) could also give additional insights in elucidating the role of methylation of the selected genes.

Conclusions
The methylation of the MLH1, MGMT, DAPK, and CASP8 gene promoters occurs in cancerous as well as noncancerous stomach tissues. An inverse correlation between the methylation of the MLH1 and DAPK promoter genes was found. Our findings provide additional insights in the puzzle of gene methylation patterns in gastric adenocarcinoma.

statement of Conflict of Interest
The authors state no conflict of interest.