Abstract
Background: Recently, Glockner et al. identified the methylation of TFPI2 as a frequent event in human colorectal cancer using a gene expression array-based strategy. Materials and Methods: Methylation status of the TFPI2 gene was examined in primary carcinomas and the corresponding normal tissues derived from 38 patients with gastric cancer using quantitative methylation-specific PCR (qMSP) and the correlation between the methylation status and the clinicopathological findings was evaluated. Results: Aberrant methylation of the TFPI2 gene was detected in 7 out of 38 (18%) primary gastric carcinomas, suggesting that the methylation of TFPI2 is frequently observed in gastric carcinomas. The clinicopathological data were correlated with the methylation results. A significant difference was observed in maximal tumour size (p=0.0084), extent of tumour (p=0.0068), and TNM stage (p=0.0392). Conclusion: TFPI2 is frequently methylated in advanced gastric carcinomas.
Accumulating evidence indicates that gastric cancer is the result of various genetic and epigenetic alterations of oncogenes, tumour suppressor genes, DNA repair genes, cell-cycle regulators, and cell adhesion molecules (1). Aberrant methylation of CpG-rich sequences (CpG islands) is an epigenetic change that is common in human cancer (2). In gastric cancer, the inactivation of human mutL homolog 1 (hMLH1), O-6-methylguanine-DNA methyltransferase (MGMT), tissue inhibitor of metalloproteinase 3 (TIMP-3) and p16 by promoter hypermethylation has been demonstrated (3-6). There has been substantial interest in attempting to adapt such cancer-associated aberrant gene methylation for clinical use.
Recently, it has been reported that TFPI2 (tissue factor pathway inhibitor-2) is frequently silenced in human hepatocellular carcinoma via epigenetic alterations, including promoter methylation and histone deacetylation (7). TFPI2 is a Kunitz-type serine proteinase inhibitor that protects the extracellular matrix of cancer cells from degradation and inhibits in vitro colony formation and proliferation (7, 8). Subsequently, Glockner et al. identified the methylation of TFPI2 as a frequent event in human colorectal cancer using a gene expression array-based strategy (9). These results prompted the present study to examine the methylation status of the TFPI2 gene in gastric tumours surgically removed.
In the present study, the methylation status of the TFPI2 gene was examined in primary carcinomas and the corresponding normal tissues derived from 38 patients with gastric carcinomas, and the correlation between the methylation status and the clinicopathological findings was evaluated.
Materials and Methods
Sample collection and DNA preparation. Thirty-eight primary tumour and corresponding normal tissue specimens were collected consecutively at Showa University Fujigaoka Hospital from gastric cancer patients during gastric surgery. All tissue specimens were confirmed histologically. Written informed consent, as required by the Institutional Review Board, was obtained from all patients. The samples were stored immediately at −80°C until analysis. DNA was prepared as described elsewhere (10). Briefly, tumour samples were digested overnight by proteinase K and treated by phenol/chloroform. DNA was precipitated by ethanol and ammonium acetate. The clinicopathological profiles of the patients enrolled in the study are shown in Table I.
Sodium bisulfite modification. One μg of the genomic DNA extracted from the tumour and the corresponding normal gastric tissue specimens was subjected to bisulfite treatment using an Epitect Bisulfite Kit (Qiagen, Hilden, Germany).
Quantitative methylation-specific PCR (qMSP). The bisulfite-treated DNA was amplified with qMSP conducted in a Thermal Cycler Dice® Real-Time System TP800 (Takara Bio Inc., Otsu, Japan). Thermocycling was performed in a final volume of 25 μl containing 1.0 μl of the DNA sample, 100 nM each of the TFPI2 or β-actin primers (forward and reverse) and 12.5 μl of SYBR Premix Ex Taq II (Takara Bio Inc.), which consists of Taq DNA polymerase, reaction buffer and deoxynucleotide triphosphate mixture. The qPCR primer sequences for TFPI2 have been described elsewhere and were: TFPI2 MS (sense), 5′-GTTCGTTGGGTAAGGCGTTC-3′, and TFPI2 MAS (antisense), 5′-CATAAAACGAACACCCGA ACCG-3′. PCR amplification consisted of 40 cycles (95°C for 5 s and 60°C for 30 s) after an initial denaturation step (95°C for 10 s). The bisulfite-treated DNA obtained from L132 cells, which were fully methylated by SssI methylase, was used as a positive control. To correct for differences in both quality and quantity between samples, β-actin was used as an internal control. The targets were obtained from the same bisulfite-treated DNA.
Clinicopathological features and TFPI2 methylation in gastric cancer.
TFPI2 methylation scores. The relative amounts of TFPI2 methylated DNA in the gastric carcinomas and the corresponding normal tissues normalised to the internal control β-actin were calculated. The TFPI2 methylation score in each tissue was defined as follows: relative amount of TFPI2 in tumour/average relative amount of TFPI2 in all corresponding normal tissues. TFPI2 methylation was considered positive when the methylation score was more than 5.0.
Statistical analysis. The associations between TFPI2 methylation and clinicopathological parameters were analysed using Chi-square tests or Student's t-tests. A p-value <0.05 indicated statistical significance.
Results
Methylation of the TFPI2 gene was detected in 7 out of the 38 (18%) primary gastric carcinomas, suggesting that the methylation of TFPI2 is frequently observed in gastric carcinomas.
The clinicopathological data were correlated with the methylation results. No significant correlations were observed between the presentation of methylation in gastric carcinomas and patient gender or age, histology, or lymph node metastasis (Table I). A significant difference was observed in maximal tumour size (p=0.0084), extent of tumor (p=0.0068) and TNM stage (p=0.0392) (Table I). These results indicated that TFPI2 is frequently methylated in advanced gastric carcinomas.
Discussion
Gastric cancer is one of the most common malignancies worldwide (11). In order to treat this fatal cancer, surgical operations and subsequent chemotherapy and radiotherapy are performed. For this purpose, it is important to identify the occurrence of genetic alterations as a new parameter to estimate the malignancy of the cancer.
In the present study, frequent methylation of TFPI2 was observed in gastric cancer. Moreover, a significant difference was observed in regard to maximal tumour size, extent of tumour and TNM stage, thus indicating that TFPI2 is frequently methylated in advanced gastric carcinomas. Previously, the methylation status of the MGMT gene in primary carcinomas and the corresponding normal tissues derived from 38 patients with gastric cancer were examined using qMSP (12). Aberrant methylation of the MGMT gene was detected in 4 out of the 38 (11%) primary gastric carcinomas. A significant difference was observed in the extent of tumour (p=0.0470), lymph node metastasis (p=0.0470) and TNM stage (p=0.0377), suggesting that MGMT is frequently methylated in advanced gastric carcinomas. Taken together, parameters such as the extent of tumour and TNM stage indicated that the methylated status of gastric carcinomas is significantly correlated with malignant potential. TFPI2 methylation could be used as a tumuor marker in clinical samples such as serum, for the detection of gastric carcinomas (13-15).
This study provides a solid basis for additional studies on the molecular mechanism of TFPI2 in gastric carcinoma and the present findings also suggest that TFPI2 may play a role in the carcinogenic pathway in some patients with gastric carcinomas. Since gastric carcinoma is one of the most aggressive of all cancer types, it may not be possible to improve the overall survival rate using information about TFPI2 methylation alone. However, TFPI2 methylation could prove useful as a marker for advanced gastric carcinoma. TFPI2 methylation should therefore be the target of future therapies for gastric carcinomas.
- Received April 14, 2010.
- Revision received May 25, 2010.
- Accepted June 3, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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