| 1 BACKGROUNDEsophageal squamous cell carcinoma (SCC) is one of the six most common malignant diseases in the world. Linzhou city (formerly Linxian) and nearby counties in Henan Province have been well documented as the highest incidence area for SCC in the world and SCC remains a leading cause of cancer-related deaths in these areas. The reasons for this stable incidence pattern are because of that: the exact etiological factors for SCC have not very clear; and there are lack of useful reagents for chemoprevention, sensitive biomarkers for early detection and high-risk subject screening. Apparently, to elucidate the molecular mechanism involved in esophageal carcinoma and to identify the molecular biomarkers for high-risk subject screening and early detection is the key strategy to decrease the mortality rate for SCC. However, the molecular mechanism for SCC is largely unknown.Recent studies indicate that, RASSF1A (Ras association domain family 1A gene) methylation, one of the new candidates of tumor suppressor genes, is a common event in many human cancers, including lung, liver and nasopharyngeal cancers. Moreover, the subjects with precancerous lesions of breast and lung with RASSF1A methylation have been identified to have higher risk to develop into cancer. Furthermore, RASISF1A methylation and its correlation with clinical pathology changes in SCC has been reported in Japan and Hong Kong people, but the results are controversy with breast and lung studies, and there is no report on RASSF1A methylation in Linzhou people from the higher incidence area for SCC.Thus, the present study was performed to characterize RASSF1A methylation and its correlation with clinical pathological changes in SCC from the patients at high-incidence area in Linzhou, Henan, and to shed light on molecular mechanisms involved in SCC.2 MATERIALS AND METHODSIn this study, 79 surgically resected SCC specimens (45 males with a mean age of 59±10 years old and 34 females with a mean age of 60±9 years old) from Yaocun Esophageal Cancer Hospital, Linzhou, Henan, China. Additionally normal esophageal tissue adjacent to cancer was collected from 20 patients with SCC (12 males with a mean age of 61±9 years old and 8 females with a mean age of 59±9 years old). All the patients did not received radiotherapy or chemotherapy before operation. All the subjects were conformed histopathologically as primary SCC clinicopathological changes, including primary tumor location, lymph node metastasis, tumor staging, gross classification(ulcerative type, exophytic type, medullary type, sclerotic type, intracavitary type) and differentiation.DNA extraction from tumor and normal tissues was performed with Gentral Company DNA Extraction Kit. CHEMICON company CpGenome? DNA Modification Kit (S7820) was used to modify DNA. Methylation special PCR (MSP) was applied to determine RASSF1A methylation. The SPSS13.0 was used for statistics, and P<0.05 was considered as significant.3 RESULTS3.1 RASSF1A methylation in SCC and its correlation with the changes in normal tissue from the same patientThe detecting rate of RASSF1A methylation in SCC was 67% (53/79), which was much higher than in the normal tissue (3/20, 15%), P <0. 05. Of the 20 cases examined for RASSF1A methylation with matched SCC and normal tissue from the patient, 3 cases were found with RASSF1Amethylation both in the normal and cancer tissues from the same patient with a positive consistent rate of 15%; 10 cases were observed with negative methylation both in the normal and cancer tissues (50%). In the 20 cases, RASSF1A methylation rate in SCC was apparently higher than inmatched normal tissue from the same patient (10/20,50% vs. 3/20,15%), P<0.05.3.2 RASSF1A methylation and its correlation with clinicopathological changes in SCCRASSF1A methylation rate was higher in poorly differention SCC (19/21, 90%) than in moderate (13/29, 45%) and well differention SCC (21/29, 72%) , P<0.05. The methylation observation was that the SCC patients with an age group of 41-59 years old had a higher RASSF1A methylation rate (31/37, 84%) than those with an age groups of -40 (1/3, 33%) and 60+ years old (21/39, 54%), P<0.05. RASSF1A methylation rates in different gross types of ulcerative type, exophytic type, medullary type, sclerotic type and intracavitary type were (10/15, 67%) , (14/22, 64%) , (13/23,57%),(10/13, 77%) and (6/6,100%) , respectively, but, the difference was not significant(P>0.05.). RASSF1A methylation was similar in tumor staging, lymph node metastasis, gender, and location, P>0.05.4 CONCLUSIONS4.1 As; higher consistent rate for RASSF1A methylation (65%) in the matched normal and cancer tissue from the same patient suggest that RASSF1A methylation is an early event in esophageal carcinogenesis, and may be a promising biomarker for high-risk subject screening.4.2 RASSF1A methylation is correlated with patient age and differentiation, suggesting that RASSF1A may be one of the prognostic indicators for SCC.
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