【作者】 王建炳；

【导师】 杨功焕； 乔友林；

【作者基本信息】 北京协和医学院 ， 流行病与卫生统计学， 2010， 博士

【摘要】 研究背景食管癌是常见的恶性肿瘤之一,2002年全球有462,000例新发食管癌病例和386,000例死亡病例。其中80%以上的食管癌发生在发展中国家,大部分是食管鳞癌。近年来大量的研究发现,吸烟和饮酒是食管癌低发区国家的主要危险因素,不是高发地区食管癌的主要决定因素。蔬菜和水果摄入不足,社会经济状况低下和ESCC家族史是高危人群主要的危险因素。牙齿缺失／口腔卫生不洁,与烟草无关的多环芳烃暴露,暴露于真菌毒素也可能是其危险因素。但这些因素并未与食管癌的高发有很强的关联性。感染因素作为肿瘤的危险因素越来越受关注,zur Hausen因为发现HPV是子宫颈癌的病因而获得诺贝尔奖。2002年,全球17.8%(190万)的肿瘤新发病例归因于各种感染因素(如乙型肝炎病毒、丙型肝炎病毒、幽门螺杆菌等),低和中等收入国家感染相关的肿瘤负担高于高收入国家。Syrjanen在1982年,提出了HPV感染与食管癌相关的研究假设,在随后20多年中,研究发现不同研究中HPV在食管鳞癌中的感染情况的变化范围很大。中国人群中有关HPV与食管癌关系的研究不多,且食管癌组织、拉网细胞和血清标本中有关HPV和食管癌的研究结果不同。在HPV与相关肿瘤(子宫颈癌、扁桃体癌等)的关系已经被明确的同时,HPV在食管癌发生发展过程中的作用仍不清楚。食管癌与HPV的关系仍然存在争议。本研究团队在林县进行了2个有关HPV与食管鳞癌的研究。一个是食管癌血清学的前瞻性研究,另一个研究是采用hc2方法检测食管拉网细胞学标本中HPV的感染情况。两个研究都提示HPV与食管癌没有关联。为了进一步证实食管癌与HPV的关联,需要进一步检测食管癌组织中HPV的感染情况和评价HPV在HPV阳性病人中致癌活性。已经明确的食管癌危险因素包括吸烟、饮酒、蔬菜摄入不足和水果摄入不足。许多高收入国家已经估计了肿瘤危险因素的人群归因危险度(PAF),而低和中等收入国家相关的研究较少。所以本论文另一部分工作是估计了2005年中国人群中吸烟、饮酒、蔬菜和水果摄入不足对食管癌的人群归因危险度。研究目的通过确定食管鳞癌中HPV的感染率、HPV阳性病人中HPV的致癌活性、比较来自不同实验室HPV感染率的检测结果进一步验证中国人群中食管鳞癌与HPV的关联。估计2005年中国人群吸烟、饮酒、蔬菜和水果摄入不足对食管癌的PAF值,为食管癌的病因学研究和防治措施的制定提供科学依据。材料和方法所有研究对象均来自在河南林县姚村卫生院进行食管切除术的食管鳞癌病例,年龄均在18岁以上,272例食管癌病例都签署了知情同意书。我们采用标准的无菌方法收集和处理食管癌组织,收集的食管癌标本包括冷冻组织和福尔马林固定的石蜡包埋的食管癌组织,食管癌冷冻组织在约翰霍普金斯大学实验室采用PGMY引物、HPV16和18特异性E6/E7引物检测HPV的感染率,而福尔马林固定的石蜡包埋的食管癌组织于中国医学科学院肿瘤研究所流行病学HPV实验室采用SPF10引物进行检测。HPV阳性的食管癌病例检测p16INK4a蛋白过表达和HPV16和18 E6/E7的血清抗体以进行致癌活性的评价。同时我们采用经典的PAF计算公式估计吸烟、饮酒、蔬菜和水果摄入不足相关的食管癌死亡和发病的负担。中国人群的吸烟、饮酒、蔬菜和水果摄入量的暴露数据来自有代表性的大样本研究。吸烟、饮酒、蔬菜和水果的摄入不足与食管癌危险的RR值来自Meta分析或大样本的前瞻性研究。食管癌死亡和发病数据来自全国第三次死因回顾调查和肿瘤登记点的数据。结果1.食管鳞癌的HPV的病因学本研究的食管鳞癌病例来自13个省、直辖市、自治区,其中27.6%的病例来自太行山地区,72.4%来自其他的地区。272例食管癌病人的中位年龄为60岁(范围：38-79岁),大约2/3为男性,约98%的人已经结婚。该人群现在吸烟率较低,只有8.1%的人目前吸烟。160(58.8%)曾经吸烟,其中2例为女性吸烟者。现在饮酒率也较低,仅为9.9%。而110例食管癌病人为曾经饮酒,全部为男性。272例食管癌病人中,102(37.5%)例病人既不吸烟也不喝酒,其中女性有83人。研究对象中67人(24.6%)有肿瘤家族史,其中55人(20.2%)有食管癌家族史。272例食管鳞癌病例中有3例HPV阳性。进一步HPV致癌活性评价试验结果显示：3例HPV阳性食管鳞癌病例没有发现p16INK4a蛋白过表达和HPV16血清E6/E7抗体阳性。2.食管癌环境危险因素的人群归因危险度研究估计2005年中国人群85,421例食管癌死亡和106,167例食管癌发病归因于4个危险因素,即吸烟、饮酒、蔬菜摄入不足和水果摄入不足。其中约24,626例食管癌死亡和30,560例食管癌发病归因于吸烟,其中男性食管癌死亡23,528例和发病29,187例(17.9%),女性食管癌死亡1,098例和发病1,373例(1.9%)。约20,749例食管癌死亡和25,748例发病病例归因于饮酒,其中男性食管癌死亡19,989例和发病24,797例(15.2%),女性食管癌死亡760例和发病951例(1.3%)。2005年中国人群蔬菜摄入量增加到最高五分位数水平,能避免8,005例食管癌死亡和9,956例食管癌发病,其中包括男性死亡5,628例和发病6,982例(4.3%),女性死亡2,377例和发病2,974例(4.1%)。水果摄入量增加到最高五分位数水平能避免52,058例食管癌死亡和64,745例食管癌发病,其中包括男性死亡35,666例和发病44,244例(27.1%),女性食管癌死亡16,392例和发病20,501例(28.0%)。结论1.该研究结果与以前林县的2个研究相似,HPV不是中国人群食管鳞癌的危险因素,HPV在食管鳞癌的致癌过程中可能不起重要的作用。2.中国人群中,大约一半的食管癌发病或死亡归因于吸烟、饮酒、蔬菜／水果摄入不足,其中约30%的食管癌发病或死亡归因于吸烟和饮酒。3.未来需要进一步的大样本、前瞻性的研究明确食管癌发病或死亡的其他危险因素。更多还原

【Abstract】 BackgroundEsophageal cancer is one of the most common cancers, with 462,000 new cases and 386,000 deaths in 2002 worldwide. More than 80% of esophageal cancers occur in the developing countries. From recent studies, smoking and alcohol drinking were found to be the major risk factors for ESCC in low risk countries, however, they were not determinant risk factors for ESCC in high risk countries. Low vegetable and fruit intake, low socioeconomic status and family history of ESCC are major risk factors for ESCC in high risk populations. Tooth loss/poor oral hygiene, polycyclic aromatic hydrocarbons (PAHs) and exposure to mycotoxin are considered to be possible risk factors for ESCC. However, none of these risk factors has shown a strong association with ESCC. Today, there is growing concerns about infectious agents as risk factors for cancer. In 2008, Dr zur Hausen was awarded the Nobel Prize for his discovery of human papilloma virus causing cervical cancer. In 2002 about 17.8% of cancers (1.9 million cases) were attributable to infectious agents (Hepatitis B and C viruses, Helicobacter pylori, etc.) worldwide. The disease burden of infection-related cancers in low-and medium-income nations is much heavier than that in high-income nations.In 1982, HPV was suggested as a risk factor for esophageal cancer by Syrjanen. And in the following two decades, HPV infection rate in esophageal cancer varied in different studies. Few studies have been conducted to explore the association between HPV and esophageal cancer in China and different results were shown when diverse samples including tissue, cytology and serum were used. Although relationship of HPV with several cancers (cervical cancer., tonsillar cancer)has been established, yet carcinogenesis of HPV in the esophagus remains unknown. Association between HPV and esophageal cancer stays controversial.We have previously conducted two studies of HPV and ESCC in Linxian, China. One was a prospective study that evaluated the association between seropositivity for HPV antibodies and the risk of having ESCC. The other was a study that detected the prevalence of HPV in.esophageal cytology specimens using Hybrid Capture 2 test. These two studies found no association between HPV and ESCC. To further study HPV in ESCC, we need to detect HPV from ESCC tissues and to evaluate the carcinogenicity of HPV in HPV positive cases.Epidemiology studies have demonstrated that smoking, alcohol drinking, low vegetable intake and low fruit intake are risk factors of esophageal cancer. Most previous studies of attributable fraction of cancers were conducted in high income countries, while few studies were carried out in low income countries. Therefore, the second part of the thesis is to estimate the proportion of esophageal cancer mortality and incidence attributable to known risk factors in China.ObjectiveTo definitively evaluate the association between HPV and ESCC by determining the prevalence of HPV in ESCC tumors, the activity of HPV in HPV positive cases and comparing HPV prevalence results from different laboratories.To estimate the proportion of esophageal cancer deaths and cases attributable to smoking, alcohol drinking,low vegetable or fruit intake and provide evidence for decision makers on esophageal cancer control in China.Materials and Methods272 consecutive incident histopathologically confirmed ESCC cases who "were≥18 years of age and undergoing surgical resection at Yaocun Commune Hospital in Linxian, China were collected from October 2006 to March 2007. All subjects provided written informed consent. Sample collection and process were based on,a standardized protocol designed to minimize the possibility of tissue contamination by environmental HPV. Tumor tissues were classified as frozen tissues and paraffin-embedded formalin fixed tissues. Frozen tissues were tested for HPV DNA using PGMY primers and HPV16/18 E6/E7 primers at Johns Hopkins University. Paraffin-embedded formalin fixed tissues were detected for HPV DNA at CICAMS using PCR with SPF10 primers (DDL Diagnostic Laboratories, Netherlands). HPV DNA positive cases were evaluated for HPV carcinogenicity using immunohistochemical analysis of p16INK4a overexpression and enzyme-linked immunosorbent assays with glutathione S-transferase fusion proteins testing for anti-E6 and anti-E7 antibodies (available for HPV types 16 and 18).We calculated the proportion of esophageal cancers attributable to tobacco smoking, alcohol drinking, low vegetable and fruit consumption. Exposed data on smoking, alcohol drinking, low vegetable and fruit intake were from large scale national surveys of representative samples from Chinese population. Data on relative risk between smoking, alcohol drinking, low vegetable and fruit intake and esophageal cancer were derived from the Meta-analyses and large scale prospective studies. Esophageal cancer mortality and incidence were originated from the third national death cause survey and cancer registries in China.Results1. Association between HPV and ESCC Patients in our study came from 13 of the 31 Provinces, Municipalities, and Autonomous Regions of China, including both the high risk Taihang mountain region (27.6%) and other areas (72.4%). Median age of 272 esophageal cancer cases was 60 years (age range:38-79 years). More than two thirds of the cases were men and nearly all were married (98%). One hundred and sixty (58.8%) of the patients were ever smokers. All but 2 of them were males. Prevalence of current alcohol drinking was low (9.9%). Similarly,110 (40.4%) of the patients were ever drinkers, all of them were male. Among 272 esophageal cancer cases,102 (37.5%) were never smokers and never, drinkers,83 of whom were female. Sixty seven patients (24.6%) had a family history of cancer, among them,55 (20.2%) had esophageal cancer family history. Among the 272 cases,3 were positive for HPV. One male case was tested weakly positive for HPV89 using PGMY consecutive primers and Roche HPV Linear Array Assay. Two cases were tested weakly positive using SPF10 primers and LiPA genotyping tests:one female for HPV 16 and one male for HPV31. Tests for HPV carcinogenicity showed that none of the 3 HPV positive cases exhibited p16INK4a protein overexpression and the HPV 16 positive case was seronegative for HPV 16 E6/E7 antibodies.2. Attributable causes of esophageal cancer in China We estimated that a total of 85,421 esophageal cancer deaths and 106,167 cases were attributable to 4 known risk factors in China in 2005, including smoking, alcohol drinking, low vegetable intake and low fruit intake. Among them,24,626 esophageal cancer deaths and 30,560 incident cases were attributable to smoking (23,528 deaths and 29,187 cases in men (17.9%) and 1,098 deaths and 1,373 cases in women (1.9%)). A total of 20,749 deaths and 25,748 cases were attributable to alcohol drinking (19,989 deaths and 24,797 cases in men (15.2%) and 760 deaths and 951 cases in women (1.3%)). Increasing consumption of vegetables to the highest quintile could avoid 8,005 esophageal cancer deaths and 9,956 cases, including 5,628 deaths and 6,982 cases among men (4.3%) and 2,377 deaths and 2,974 cases among women (4.1%). Increasing consumption of fruit to the highest quintile could avoid 52,058 esophageal cancer deaths and 64,745 cases, including 35,666 deaths and 44,244 cases among men (27.1%) and 16,392 deaths and 20,501 cases among women (28.0%).Conclusions1. The result of our study is similar to the two previous studies of HPV and ESCC in Linxian. HPV is not a risk factor of esophageal cancer in Chinese population. HPV is not involved in esophageal carcinogenesis.2. In Chinese population, about half of esophageal cancer deaths or cases are attributable to smoking, alcohol drinking, low vegetable intake and low fruit intake. Smoking and alcohol drinking are responsible for about 30% of esophageal cancer mortality or incidence.3. We need to conduct large scale, multicenter studies to demonstrate other important risk factors of esophageal cancer.更多还原