RASSF1A Gene Methylation At Promotor Region And Its Correlation With Gross Morphological Changes In Gastric Cardia Adenocarcinoma

1 BACKGROUNDGastric cardia adenocarcinoma (GCA) is one of the most common malignant digestive diseases in northern China. In contrast to the strikingly decline of incidence of distal gastric cancer around the world in the past two decades, the incidence of GCA, especially in America and Europe, has increased dramatically, with increasing speed of 20% annually, which is one of the fastest increasing malignant diseases. The reason is not clear yet. Furthermore GCA is an under studied subject in China, the information for molecular changes related with GCA is very limited.Recent studies by us and other laboratory demonstrate that: Ras association domain family A gene (RASSF1A), one of the new candidates of tumor suppressor genes, has been methylated in many different human cancers, including lung, nasopharyngeal, liver and gastric cardia cancers. An interesting observation is that the data for RASSF1A methylation is controversy in different laboratory reports. Our hypothesis is that different gross types of GCA many have different methylation condition. Our recent studies have demonstrated higher RASSFIA methylation in GCA. Thus, the present study was undertaken to determine RASSFIA methylation patterns based on GCA gross types and to correlate the changes of RASSFIA methylation with clinicopathological changes, including tumor staging.2 MATERIALS AND METHODSIn this study, 81 surgically resected GCA specimens (58 males and 23 females,with a mean age of 60±8 years old) from Yaocun Esophageal Cancer Hospital, Linzhou, Henan, China. Normal gastric cardia tissue adjacent to cancer was collected from 20 patients with GCA. All the patients did not received radiotherapy or chemotherapy before operation. All the specimens were identified by pathological examination to four gross types. Gross classification for GCA specimens was made based on the criteria in China, of the 81 GCA specimens, there were 32 with infiltrative type, 18 with local ulcerative type, 17 with exophytic type, 14 with infiltrative ulcerative type.DNA extraction from tumor and normal tissues was performed with Gentral Company DNA Extraction Kit. CHEMICON company CpGenome^TMDNA Modification Kit (S7820) was used to modify DNA. Methylation special PCR (MSP) was applied to determine RASSF1A methylation. The x² tests were used for statistics, and P<0.05 was considered as significance.3 RESULTS3.1 RASSFIA methylation in GCA was 72% (58/81), which was much higher than in the normal tissue (15% , 1/20). the difference was significant, P<0.01.3.2 RASSFIA methylation and its correlation with gross classification changes in GCA. The proportion of four gross types were: infiltrative ulcerative (79%, 11/14), local ulcerative (78%, 14/18), infiltrative (69%, 22/32), exophytic (65%, 11/17) respectively. The differences were not observed among these groups, P >0.05.3.3 RASSFIA methylation and its correlation with clinicopathological changes in GCA. The rate of RASSFIA gene promotor methylation in male and female were 68% (36/53) and 79% (22/28), respectively. The difference was not significant, (P>0.05). RASSFIA methylation rate was observed in 29 in young and mediator patients (78%), and 29 in old patients (66%). The differences among the two groups were not significant, (P>0.05). The methylation rate of RASSFIA gene in poorly, moderate and well differentiated groups were 63%, 83% and 73% respectively. There were no significant differences among these groups, (P>0.05). The methylation rate of RASSFIA gene in moderately stage and late stage groups were 68% and 74% respectively. There were no significant differences among these groups, (P>0.05).4 CONCLUSIONS4.1 As higher consistent rate for RASSFIA methylation (72%) in the matched normal and cancer tissue from the same patient suggest that RASSFIA methylation is a frequent event in GCA.4.2 The frequency of RASSFIA methylation in different gross types of GCA is similar, which is not consistent with our hypothesis.4.3 There is no certain relationship between clinicopathological changes and RASSF1A methylation in GCA.