【作者】 吴会芳；

【导师】 王立东；

【作者基本信息】 郑州大学 ， 内科学， 2007， 硕士

【摘要】 1．背景中国北方太行山区是食管鳞癌(Esophageal squamous cell carcinoma，SCC)和贲门腺癌(Gastric cardia adenocarcinoma，GCA)同时高发的地区。SCC和GCA好发年龄段均为50岁以上的中老年人。近年的研究提示，中国北方SCC高发区青年(=40岁)食管鳞癌和贲门腺癌并不罕见。某些学者认为：与中老年SCC患者相比，青年SCC患者癌细胞分化差，易发生浸润性生长和早期血管转移，5年生存率更低，预后极差。提示青年SCC和GCA可能有其独特的分子基础。青年患者的存在引发一个重要的学术问题：为什么在相同的生活环境中，青年患者可提前10余年发病?我们的假设是：个体的遗传易感因素可能在青年SCC和GCA发病中起更重要的作用。河南SCC和GCA高发区存在明显的SCC和GCA家族聚集现象，进一步提示遗传因素在SCC和GCA癌变中可能起重要作用。但SCC和GCA遗传易感性的分子机制尚不十分清楚。近年研究提示，RASSF1A(Ras相关区域家族1A，Ras-association domain family1A)基因是2000年克隆得到的新的肿瘤抑制基因，位于染色体3p21.3区。它所表达的产物广泛存在于肺、肝脏、消化道等多种器官和组织中，在调控细胞的增殖和凋亡中具有重要作用，体内外均能抑制肿瘤的生长。RASSF1A启动子甲基化是其失活的主要机制。但是，目前尚未见到RASSF1A基因与中国北方太行山高发区青年SCC和GCA癌变关系的报道。本研究对中国北方太行山高发区SCC和GCA高发区14814例SCC患者和7457例GCA患者进行回顾性调查，了解SCC和GCA高发区青年SCC和GCA的检出率及临床病理特点；检测部分青年SCC和GCA组织中RASSF1A启动子甲基化及蛋白表达，探讨其在SCC和GCA癌变中的作用，以加深对青年SCC和GCA遗传易感性的认识。2．材料与方法2．1材料来源所有临床病理资料均来自1974年-2006年山西省磁县人民医院和姚村食管癌医院以及1998年-2006年林州中心医院14814例SCC和7457例GCA的临床和病理记录，根据病理诊断报告和住院记录分别统计SCC患者人数、性别、年龄、病理特征等，并对这些数据进行统计分析。SCC和GCA组织标本均来自林州地区患者手术切除标本。SCC共40例，其中青年组患者13例，平均年龄39±1岁；老年组患者27例，平均年龄72±2岁。GCA共24例，其中青年组患者10例，平均年龄37±2岁；老年组患者14例，平均年龄68±3岁。食管和贲门正常组织均取自同一个体癌旁正常组织(组织病理学证实)。所有患者术前均未接受任何放疗或化疗。2．2组织处理手术切除标本一半经85％酒精固定、石蜡包埋、连续切片，进行组织病理学检查，一半立即置-80℃低温冰箱中，用于冰冻切片和DNA提取。2．3试验方法2．3．1免疫组织化学染色采用冰冻切片免疫组织化学ABC法检测RASSF1A蛋白表达，鼠抗人RASSF1A单克隆抗体(美国eBioscience公司)工作液浓度为原液的1／500。2．3．2甲基化特异性PCR对于RASSF1A基因启动子区甲基化水平的检测采用1996年Herman等提出的甲基化特异性PCR方法(methylation-specific polymerase chain reaction，MSP)。原理：DNA在亚硫酸氢盐的作用下，未甲基化的胞嘧啶(C)发生脱氨基反应转变成了尿嘧啶(U)，发生甲基化的5-甲基胞嘧啶(5-~mC)不发生转变，因此处理后的DNA序列将出现不同的情况，基于这两种情况设计了甲基化与非甲基化特异性引物。对同一例修饰后的DNA分别用甲基化与非甲基化特异性引物扩增，二者将至少有一个扩增。根据上面的原理，就可以由特异性引物判断原来DNA的甲基化状态。2．4统计学方法采用SPSS13.0统计软件处理，各组间的比较采用χ~2检验，一致性分析采用Kappa检验，显著性水准取α=0.05。3．结果3．1青年SCC的检出率及临床病理特征3．1．1三家医院14814例SCC患者中共有青年SCC患者625例，占同期SCC患者的4％。但三家医院青年患者占同期SCC患者的比例并不相同。磁县人民医院最高(8％)，分别是林州中心医院(2％)和姚村食管癌医院(4％)的4倍和2倍，差异显著(P＜0.05)。3．1．2同一家医院不同年份青年SCC患者占同期SCC患者的比例并不相同。姚村食管癌医院1976-1997的比例(7％)显著高于1998-2006年(2％)(P＜0.05)；磁县人民医院1974-1997的比例(9％)是1998-2006年(3％)的3倍(P＜0.05)。3．1．3 625例青年SCC患者男女比例为1.72，与中老年SCC患者男女比例1.71相似(P＞0.05)。3．1．4青年(24％)与中老年(26％)患者淋巴结发生转移的比例相似(P＞0.05)。其中男性淋巴结发生转移的比例高于女性(青年：25％vs．21％；中老年27％vs．25％；P＜0.05)。青年SCC高分化的比例高于中老年(10％vs．7％；P＞0.05)。青年SCC浸润纤维膜的比例低于中老年患者(P＜0.05)。3．2青年GCA的检出率等流行病学特征3．2．1三家医院33年7457例GCA患者中共有青年GCA患者196例，占同期GCA患者的3％。青年GCA的检出率三家医院相似。3．2．2青年GCA患者男女比值为2.84，低于中老年GCA患者男女比例3.36(5594／1667)，但差异无统计学意义(P＞0.05)。3．3三家医院SCC和GCA流行特征比较3．3．1青年GCA占青年SCC和GCA的比值为24％，低于中老年GCA的这一比值(33％)，差异有统计学意义(P＜0.05)。磁县人民医院青年GCA在SCC和GCA中的构成比最低(18％)，中心医院最高30％(P=0.011)；磁县人民医院总的GCA在SCC和GCA中的构成比(36％)高于姚村食管癌医院(34％)(P＜0.05)。3．4 RASSF1A蛋白表达3．4．1青年SCC组织RASSF1A蛋白表达高于老年，差异显著(83％，40％，P＜0.05)；同样的情况见于青年GCA组织(89％，36％，P＜0.05)。3．4．2 RASSF1A在青年SCC和GCA组织中阳性表达频率均较高(83％，10／12vs 89％，8／9)，与对应正常组织中的表达差异没有统计学意义(P＞0.05)，但表达类型并不相同。3．4．3老年SCC组织RASSF1A蛋白表达低于食管正常上皮组织(40％，77％，P＞0.05)；同样的情况见于老年GCA组织和正常上皮组织，后者差异显著(36％，91％，P＜0.05)。3．4．4 SCC和GCA组织RASSF1A蛋白阳性表达率相似(P＞0.05)。3．5 RASSF1A基因启动子甲基化3．5．1青年SCC组织RASSF1A基因启动子甲基化率高于老年，但差异无统计学意义(46％，15％)。同样的情况见于青年和老年GCA组织(60％，30％)。3．5．2青年(60％，40％)和老年(30％，29％)GCA组织RASSF1A基因启动子甲基化率稍高于正常上皮组织，但差异无统计学意义；同样的情况见于青年SCC和正常上皮组织(46％，33％，P＞0.05)。3．6 RASSF1A基因启动子甲基化与RASSF1A蛋白表达的关系3．6．1 RASSF1A基因启动子发生甲基化的19例组织中，仅有2例(11％)出现RASSF1A蛋白阴性表达；RASSF1A基因启动子未发生甲基化的44例组织中，有21例(52％)出现RASSF1A蛋白阴性表达。3．7 RASSF1A基因启动子甲基化和RASSF1A蛋白表达与患者临床病理特征的关系。3．7．1青年与老年SCC和GCA组织RASSF1A基因启动子甲基化率与淋巴结转移，分化程度及浸润深度均没有相关性。3．7．2青年与老年SCC和GCA组织RASSF1A蛋白的阳性表达率与淋巴结转移，分化程度及浸润深度均没有相关性。4．结论4．1华北太行山高发区青年SCC和GCA并不罕见(青年SCC：2％-9％；青年GCA：2％-3％)，青年SCC的组织病理特征，包括淋巴结转移，浸润深度和分化程度与中老年SCC患者相似。4．2青年SCC和GCA组织RASSF1A蛋白表达高于老年，提示青年与老年SCC和GCA具有不同的分子基础，RASSF1A蛋白阳性表达是高发区青年SCC和GCA组织中常见的分子事件。4．3青年SCC和GCARASSF1A启动子甲基化率分别为46％和60％，提示，RASSF1A启动子甲基化是青年SCC和GCA频发的分子事件，可能在青年SCC和GCA癌变中具有重要的作用。4．4 RASSF1A启动子甲基化与其蛋白表达间没有明显的相关性，提示除外启动子甲基化，可能还存在影响RASSF1A蛋白表达的其它机制，另外，RASSF1A基因启动子甲基化及蛋白表达与SCC和GCA的临床病理特征没有相关性。更多还原

【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.更多还原