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The Promoter Methylation and Protein Expression of RASSF1A Gene in the Young Patients with Esophageal Squamous Cell Carcinoma and Gastric Cardia Adenocarcinoma

Author: WuHuiFang
Tutor: WangLiDong
School: Zhengzhou University
Course: Internal Medicine
Keywords: esophageal squamous cell carcinoma gastric cardia adenocarcinoma RASSF1A methylation immunohistochemical staining young and old patients
CLC: R735
Type: Master's thesis
Year: 2007
Downloads: 83
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Abstract


1. BACKGROUNDThe Taihang mountainous area in northern China is the high incidence area for esophageal squamous cell carcinoma (SCC) and gastric cardia adenocarcinoma (GCA). Although SCC and GCA usually occurs in elderly peoples (=50 years old), the young SCC and GCA patients (=40 years old) have been frequently identified in Taihang mountainous area of northern China. Young SCC and GCA patients have been characterized with more poorly differentiated pathological type and a poor prognosis because of early diffuse infiltrative growth and vessel invasion, so the 5-year survival rate is lower than the elderly counterpart, which suggests that different molecular basis may exist .in young patients These phenomena raise an important academic question: what is the mechanism to push the youth to develop SCC and GCA ten years earlier than the old even with the same life-style and similar environment? Our hypothesis is that individual genetic susceptibility may play more important role in the development of SCC and GCA for the young patients. Present study found that there is obvious prevalence for familial SCC and GCA in high-incidence area in Henan. This also indicates the importance of genetic factors in the development of SCC and GCA. However, the information for the molecular changes is very limited in young SCC and GCA patients, and the molecular basis for the high susceptibility of the young patients is largely unknown.Recent studies suggest that Ras association domain family 1A gene (RASSF1A) is one of the new candidates of tumor suppressor genes located at 3p21.3. This gene is silenced and frequently inactivated by promoter region hypermethylation in many adult and childhood cancers, including lung, breast, kidney, gastric, bladder, etc. RASSF1A inhibits tumor growth both in vitro and in vivo systems. Thus far, it has been shown to play important roles in cell cycle regulation, apoptosis, and microtubule stability.However the alteration of RASSF1A gene in young SCC and GCA carcinogenesis has not been characterized. Furthermore there are many case reports for young patients with SCC and GCA, the information for the prevalence of SCC and GCA in young population is very limited. The present study was undertake to retrospect 14814 cases SCC and 7457 cases GCA patients from high-incidence of Taihang mountainous area in northern China for the prevalence of young SCC and GCA, and to characterize the alteration of RASSF1A methylation and protein expression of young SCC and GCA patients, to further understand the molecular mechanism of high susceptibility of the young patients.2. MATERIALS AND METHODS2.1 Tissue collection and processingAll the clinical and pathologic information of 14814 cases with SCC and 7457 cases with GCA was obtained from Linzhou City Hospital, Yaocun Esophageal Cancer Hospital and Cixian People’s Hospital, the high-incidence areas for SCC and GCA in northern China from 1974 to 2006. The clinicopathological data of SCC and GCA patients, including number of cases, gender, age and tumor staging, etc. were separately computed. A total of 64 surgically resected primary SCC and GCA were collected from the patients in Linzhou city. Out of the 64 speciments examined, 13 were from young SCC patients (36-40 years of age with a mean±SD of 39±1) and 27 old SCC patients (70-78 years of age with a mean±SD of 72±2 years old); 10 young GCA patients (34-40 years of age with a mean±SD of 37±2) and 14 old patients (65-72 years of age with a mean±SD of 68±3). The corresponding normal controls were gained from the adjacent cancer of the same patient (approved by pathology). All the patients had not received either chemotherapy or radiotherapy before surgery.2.2 Tissue processingHalf of the surgically resected specimen was placed in -80°C refrigerator and the otheir half was fixed with 85% ethanol, paraffin embedded, and serially sectioned for histopathological diagnosis.2.3 Methods23.1 Immunohistochemical stainingTo determine the protein expression of RASSF1A gene immunohistochemical staining (ABC method) was applied. Affinity purified anti-human RASSF1A (eBioscience) was used with a diluted solution of the primary antybody (1 : 500).23.2 Methylation-specific PCR (MSP)To determine the promoter methylation of RASSF1A gene, MSP was applied, which had been reported by Herman in 1996. In brief, all the cytosines are converted to uracil by bisulfite treatment, but those that are methylated (5-methylcytosine) are resistant to this modification and remain as cytosine. This altered DNA can then be amplified and sequenced. We designed primers to distinguish methylated from unmethylated DNA in bisulfite-modified DNA, taking advantage of the sequence differences resulting from bisulfite modification.2.4 Statistic analysisThe data was analyzed by SPSS13.0 statistical software. The x~2 test, Fisher’s exact test, Kappa test were used for the statistics (P<0.05 was considered significant).3. RESULTS 3.1 The prevalence and clinicopathological features of young SCC patients3.1.1 Of the 14814 SCC patients from the three hospitals, 625 young patients were identified with SCC. The proportion of the young to all the SCC in the same period was 4%. However the proportions of the three hospitals were different. Cixian People’s Hospital was the highest, 4 folds of Linzhou Central Hospital and 2 folds of Yaocun Esophageal Cancer Hospital (P<0.05).3.1.2 The proportion of the young SCC patients was different in different periods. Higlier proportion was observed in 1976-1997 (7%) than that in 1998-2006 (2%) in Yaocun Esophageal Cancer Hospital; the proportion in 1974-1997 (9%) was 3 folds higher than that in 1998-2006 (3%) in Cixian People’s Hospital.3.1.3 Of the 625 young SCC patients, the rate of young male to female(1.72 : 1) was similar with that of the elderly (1.71 :1)(P>0.05).3.1.4 The rate of lymph node metastasis in young group was 24%, which was similar with that in elderly group (P>0.05). However, the rate of lymph node metastasis in male group was higher than that in female (young: 25% vs 21%; elderly: 27% vs. 25%; P<0.05). Moderately differentiated pathological type was dominantly observed in young and elderly. However, the proportion of poor differentiated type in young patients was a little lower than that in elderly patients (12% vs. 18%, P>0.05). There was no significant difference between the two groups in the degrees of invasions. However, the proportion of invasion to involucra in young patients was a little lower than that in elderly patients (P<0.05).3.2 The prevalence of young GCA patients.3.2.1 Of the 7457 cases GCA patients from the three hospitals, 196 cases young patients were identified with GCA. The proportion of the young to all the GCA in the same period was 3%. The proportions of the three hospitals were similar.3.2.2 The rate of young male to female patients (2.84 : 1) was lower than that of the elderly (3.36 : 1), however, the difference was not significant (P>0.05).3.3 The comparison on the prevalence of SCC and GCA in the three hospitals3.3.1 The proportion of young GCA in young SCC and GCA was 24%, which was lower than that of elderly patients (33%). However, the proportions of the three hospitals were different. The proportion (36%) of elderly GCA patients in Cixian People’s Hospital was highest, and that was lowest in Y Yaocun Esophageal Cancer Hospital (P<0.05). But, the proportion of young GCA in Cixian People’s Hospital (18%) was much lower than in Linzhou Central Hospital (30%) (P<0.05).3.4 The protein expression of RASSF1A3.4.1 The positive immunostaining rate for RASSF1A in young SCC and GCA was higher than that in old SCC and GCA (esophageal: 83%, 40%, P<0.05; gastric cardia: 89%,36%,P<0.05).3.4.2 The positive immunostaining rate for RASSF1A in young SCC and GCA was 83% (10/12) and 89% (8/9), respectively, which was similar as in corresponding normal tissues (P>0.05). However, the immunostaining patterns were different.3.4.3 The positive immunostaining rate for RASSF1A in SCC and GCA from old patients was lower than that in corresponding normal tissues (esophageal: 40% vs. 77%, P>0.05; gastric cardia: 36%, 91%, P<0.05).3.4.4 The positive immunostaining rate for RASSF1A in SCC and GCA was similar (P>0.05).3.5 The promoter methylation of RASSF1A gene3.5.1 The rate of promoter methylation of RASSF1A in young SCC seemed higher than that in old SCC patients (46%, 15%).But, the difference was not significant. The similar results were observed in young and old GCA patients.3.5.2 The rate of promoter methylation of RASSF1A in GCA was a little higher than that in corresponding normal tissues (young patients: 60%, 40%; old patients: 30%, 29%). But, the difference was not significant. The similar result was observed in young SCC patients (46%, 33%, P>0.05).3.6 Association between the RASSF1A gene promoter methylation and its protein expiession3.6.1. Of the 19 cases with promoter methylation, only 2 cases (11%) were identified with negative expression of RASSF1A protein. Of the 44 cases with promoter unmethylation, there were 21 cases (52%) with negative expression of RASSF1A protein. 3.7 The association between the clinicopathological features of SCC patients and the promoter methylation and protein expression of RASSF1A gene3.7.1 There was no significant relationship between the promoter methylation of RASSF1A gene and the clinicopathological changes of SCC and GCA patients.3.7.2 There was no significant relationship between the protein expression of RASSF1A gene and the clinicopathological changes of SCC and GCA patients.4. CONCLUSIONS4.1 The present study demonstrates that the occurrence of young SCC and GCA patients is not uncommon (2%-9% for SCC and 2%-3% for GCA), and that the histopathological changes including differentiations, lymph node metastasis and invasion, were similar between the young and elderly patients with SCC.4.2 The positive immunostaining rate for RASSF1A in young SCC and GCA is higher than that in old SCC and GCA, suggesting that there may be different molecular basis involved in SCC and GCA from the young and old patients. The positive expression for RASSF1A protein is a frequent molecular event in young SCC and GCA.4.3 Promoter methylation of RASSF1A occurs in 46% of SCC and 60% of GCA from the young patients, suggesting that RASSF1A gene methylation is a frequent molecular event in young SCC and GCA, and may play an important role in oncogenesis of SCC and GCA from the young patients.4.4 There is no significant relationship between the promoter methylation and protein expression of RASSF1A gene, suggesting that there may be other mechanisms involved in the protein expression of RASSF1A. RASSF1A methylation and protein expression do not shown any correlation with the clinicopathological changes of SCC and GCA both in young and old patients.

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