【作者】 曹芳丽；

【导师】 程玉峰；

【作者基本信息】 山东大学 ， 临床医学（专业学位）， 2014， 博士

【摘要】 研究背景及意义食管癌在全球最常见的恶性肿瘤中位居第八,是癌症相关死亡的第六大原因,发病率正在迅速增加。在很多西欧国家和北美食管癌的病理类型以腺癌为主,但食管鳞状细胞癌(esophageal squamous cell carcinoma, ESCC)仍然是全球最常见的病理类型。我国位于著名的“亚洲食管癌地区带”内,食管癌的发病率和死亡率一直位居世界首位,最新的统计数据提示食管癌的发病率居我国各类恶性肿瘤第五位,死亡率高居肿瘤相关死亡的第四位,无论发病还是死亡人数均占全球一半以上,其中90%以上的食管癌病理类型是鳞状细胞癌。由于缺乏早期症状,加之肿瘤侵袭性强,大部分病人确诊时已属中晚期,预后极差。食管癌的发生是一个漫长、多因素、多步骤的过程,其病因尚不清楚。流行病学的研究资料表明：缺乏微量元素、大量饮酒、吸烟、遗传因素、不良饮食习性、慢性刺激和炎症与微生物感染等因素均参与食管癌的发生发展过程。直接或间接的病毒感染引起越来越多恶性肿瘤的发生。人类乳头状瘤病毒(human papillomavirus, HPV)是目前世界各地最常见的性传播病毒,HPV是一种小的双链脱氧核糖核酸(deoxyribonucleic acid, DNA)病毒,对鳞状上皮具有趋向性,持续的HPV感染可导致感染部位发生过度增殖性病变,进而导致癌变。这种具有潜在致癌能力的HPV被称为高危型HPV (high-risk human papillomavirus, HR-HPV)。世界上每年超过55万的新发恶性肿瘤患者与高危型HPV感染有关。HPV感染与生殖器肿瘤,尤其是宫颈鳞癌之间的关系已经建立。HPV感染与非生殖器肿瘤的关系成为各国学者研究的热点,在肿瘤组织中,高危型HPV16、18型是最广泛的感染类型,其他亚型的HPV感染极为罕见。随着阴道-口腔-肛门等性行为的多样化及性伴侣数目的增多,HPV相关性口咽部肿瘤的发病率明显升高。口咽与食管的鳞状上皮具有组织连续性和相似性,HPV感染早在1982年就被Syrjanen等人提出是食管癌的诱因之一。随着HPV在食管鳞癌发生中关键性地位的确立,其具体致癌机制目前仍不明确,目前研究较为充分的是病毒癌基因E6和E7,认为癌基因E6和E7及其产物在癌变过程中起关键作用。病毒DNA整合进入宿主细胞DNA内,病毒癌基因E6和E7激活并持续表达,其产物分别引起P53-Rb通路的功能异常,E2F的释放,进而激活细胞周期中S相导致肿瘤DNA的合成。P16基因是一个近年来发现的抑癌基因,直接作用于细胞周期,抑制细胞的分裂,其产物p16INK4a蛋白(以下简称为p16蛋白)控制细胞的生长和分化,对细胞周期进行负调控,主要抑制CDK4/CDK6蛋白的功能,CDK4/CDK6蛋白在细胞G1-S期转变过程中起关键作用。P16基因也是P53-Rb系统的重要成员,Rb基因的磷酸化,导致组织中原本异常的p16蛋白反馈性高表达,最终导致了细胞转化时检查点和调控途径的失活。致使正常食管鳞状上皮细胞的细胞周期发生紊乱,使其具有永生性,从而启动一系列的癌变过程。HPV感染鳞状上皮后p16蛋白会出现过度表达,在其他HPV相关的鳞癌组织中p16蛋白免疫组化染色早已被认定为一个客观的生物标志物,在临床检验或生物医学研究中,HPV-DNA的检测方法主要应用原位杂交(in situ hybridization, ISH)或聚合酶链反应(polymerase chain reaction, PCR),但是这两种检测方法价格相对昂贵,技术要求高,需要先进的实验室设施和经验丰富的技术人员。而免疫组化技术特异性强、敏感度高、定位准确,且操作简单易行、价格低廉,对实验设备要求不高。本课题研究食管鳞癌中HPV感染情况、HPV感染后导致相关蛋白的变化及HPV感染与食管鳞癌患者预后的关系,目的是了解HPV感染在食管鳞癌发生发展中的作用及病毒感染对食管鳞癌患者预后的影响,探索食管鳞癌中p16蛋白免疫组化染色作为HPV感染的分子替代标记物的可行性。有可能为食管癌的防治提供新的理论依据。研究分为两个部分。第一部分食管鳞癌中HPV感染与其预后的关系研究目的：HPV感染在食管鳞癌的发生发展中发挥重要作用,本部分研究旨在探讨HPV感染在食管鳞状细胞癌预后方面的价值。研究方法：共有105例行食管鳞癌根治术的患者纳入本研究,所有病例均应用DNA-DNA原位杂交技术分别检测HPV16型和18型。根据回顾性资料分析,统计患者的5年生存期和5年无疾病进展生存期,应用Cox比例风险模型进行单因素分析和多因素分析,确定影响预后的独立影响因子。研究结果：进入本研究的105例病人中男性：84例,女性：21例,中位年龄60岁(范围42岁-78岁),中位随访时间42.4月(范围4月-61月),5年随访资料完整。105例食管鳞癌患者中29例(27.6%)被检测到HPV阳性,所有阳性病例均为HPV-16型感染,无HPV-18阳性患者。HPV阳性组和阴性组的患者在分化程度、TNM分期、年龄、肿瘤部位、性别、辅助治疗及吸烟和饮酒史等临床资料方面没有统计学差异。HPV阳性组的5年生存率和5年无进展生存率是65.9%和61.8%,而HPV阴性组的5年生存率和5年无进展生存率是43.3%和36.8%,2组病人在总生存期和无疾病进展生存期方面存在统计学差异。5年生存率的Cox单因素和多因素分析显示,HPV阳性／阴性风险比分别为0.31(95%CI：0.14-0.68；P=0.004)和0.37(95%CI：0.16-0.82；P=0.01)。5年无进展生存率的Cox单因素和多因素分析显示,HPV阳性／阴性风险比分别为0.33(95%CI：0.16-0.67；P=0.002)和0.38(95%CI：0.18-0.77；P=0.008)。HPV阳性组病人的5年生存率好于HPV阴性组(P=0.004),HPV阳性组病人的5年无进展生存率好于HPV阴性组(P=0.002),HPV感染,吸烟,原发肿瘤浸润,淋巴结转移和TNM分期是影响食管鳞癌术后患者独立的预后因素。研究结论：HPV病毒感染可能是食管鳞癌发生发展的感染性因素之一,肿瘤中HPV感染是影响食管鳞癌患者的独立预后因素之一。第二部分P16INK4a蛋白在HPV相关食管鳞癌中的表达及意义研究目的：HPV感染鳞癌组织后p16INK4a蛋白(以下简称p16蛋白)会出现过度表达,本部分旨在通过研究食管鳞癌中p16蛋白表达与HPV感染之间的关系,探索食管鳞癌中p16蛋白免疫组化染色作为HPV感染的分子替代标记物的可行性。研究方法：共有105例食管鳞癌根治手术后病人纳入本部分的研究,所有病例均应用免疫组织化学方法检测p16蛋白的表达,其结果由病理学专业医师进行评估,应用Kappa检验,明确p16蛋白异常表达与HPV感染是否存在一致性。根据回顾性资料,以p16蛋白为分层因素,应用Kaplan-Meier分析统计患者的预后。研究结果：进入本研究的105例病人中男性：84例,女性：21例,中位年龄60岁(范围42岁-78岁),中位随访时间42.4月(范围4月-61月),5年随访资料完整。105例食管鳞癌患者中39例(37.1%)被检测到p16蛋白阳性表达。P16阳性组和阴性组的患者在性别、年龄、肿瘤部位、TNM分期、分化程度、辅助治疗及吸烟和饮酒史等临床资料方面没有统计学差异。29例HPV阳性的肿瘤组织中86.2%(25例)检测到p16蛋白表达阳性,76例HPV阴性的病例中18.4%(14例)检测为p16蛋白表达阳性,具有显著地统计学意义(P<0.001)。Kappa检验后,其Cohen’s Kappa系数为0.61,说明二者的表达具有很好一致性。P16阳性组的5年生存率和5年无进展生存率分别是64.1%和58.7%,而p16阴性组的5年生存率和5年无进展生存率分别是45.5%和37.9%,2组患者在总生存期和无疾病进展生存期二个方面存在明显差异。p16阳性组病人的5年生存率好于p16阴性组(P=0.02),p16阳性组病人的5年无进展生存率好于p16阴性组(P=0.007),以p16蛋白作为分层因素统计患者的预后结果与基于HPV的预后结果具有一致性。研究结论：HPV感染食管鳞状上皮后p16蛋白会出现过度表达,p16蛋白免疫组化染色可以作为HPV感染的分子替代标记物。结论：HPV感染在食管鳞癌发生发展中起重要作用,p16蛋白免疫组化染色可以作为HPV感染的分子替代标记物,肿瘤中HPV感染是食管鳞癌患者独立的预后影响因素之一。HPV感染可能成为食管鳞癌新的治疗靶点。更多还原

【Abstract】 Esophageal carcinoma is the eighth most common cancer and the sixth most common cause of death from cancer, worldwide. Despite increasing rates of esophageal adenocarcinoma in many western countries, esophageal squamous cell carcinoma (ESCC) remains the dominant histological type of esophageal cancer worldwide. China is located in the famous "Asian esophageal cancer belt", the morbidity and mortality of esophageal cancer in China has been ranked first in the world. Esophageal carcinoma is the fifth most common cancer and the fourth most common cause of death from cancer in China, and more than90%of cases are ESCC. Once developed, esophageal cancer usually rapidly invades surrounding tissues and lymph nodes. Due to the absence of early symptoms, invasiveness of the disease, and its late diagnosis, it is generally associated with a poor prognosis. The etiology of ESCC remains unclear, and epidemiological studies suggest that tobacco smoking, heavy alcohol drinking, micronutrient deficiency, and dietary carcinogen exposure may cause the malignancy. Infectious agents have been implicated, as either direct carcinogens or promoters. In particular, human papillomavirus (HPV) has been postulated as a possible cause of ESCC. HPV is currently one of the most common sexually transmitted infections worldwide. HPV is a small, nonenveloped double-stranded DNA virus with tropism for the squamous epithelium where it can cause hyperproliferative lesions, and then cause carcinogenesis. HPV with potential for oncogenesis based on persistent infection is known as the high risk HPV. The association between infection of HPV and genital cancers, especially cervical cancer has been well established, the role of HPV in nongenital cancers has become the hotspot of researchers in the world. According to previous studies. HPV-16is the most prevalent type in squamous cell carcinoma, followed by HPV-18. while other high-risk HPV types are rare. Increased numbers of vaginal, oral, and oral-anal sex partners have been associated with an increased risk of developing HPV-associated oropharyngeal cancer. Because the histological similarities between the oropharyngeal squamous epithelia and esophagus HPV infection in esophageal cancer was first suggested in1982based on histological observations by Syrjanen. The etiological role of HPV in ESCC is still unclear. Once HPV infection has taken place, viral DNA synthesis occurs. Additional accumulating changes can lead to transformation. These changes are a multistage complex interaction of the host immune system in combination with the expression of two viral oncogenes:E6and E7, followed by a series of epigenetic changes occurring in dysplastic lesions. The viral oncogene products E6and E7play a key role in HPV-associated carcinogenesis, abrogating p53and retinoblastoma tumor suppressor functions, respectively. E7binds to and degrades Rb, releasing E2F, leading to p16INK4a overexpression, hereafter denoted as p16, which is associated with superior clinical outcome. Thus HPV-positive tumors are characterized by high expression of p16and p16is widely considered a surrogate marker for HPV infection in the context of squamous cell carcinoma. Estimates of the prevalence of HPV in esophageal tumors range widely, from real-time polymerase chain reaction assays to type-specific DNA in-situ hybridization. The price of these two kinds of detection method is relatively expensive, high technical requirements, the need for advanced laboratory facilities and experienced technical staff, so restrict their wide detection application. Immunohistochemical technique is operated simply, the price is lower, is not so higher on technical requirements, Can be widely popularized. With the present study, we aim to determine the prevalence of HPV infection in ESCC, determine expression of HPV-associated proteins, and evaluate its clinical significance. We also sought to evaluate the effect of tumor HPV status on survival of patients with ESCC and want to confirm p16immunohistochemistry is a very good surrogate marker of HPV infection for ESCC. The study was divided into two parts.Part1HPV infection in ESCC and its relationship to the prognosis of patientsObjective:Human papillomavirus (HPV) as a risk factor for esophageal squamous cell carcinoma (ESCC) has previously been studied, but importance of HPV status in ESCC for prognosis is less clear.Methods:A total of105patients who underwent esophagectomy for ESCC were included in this study. All specimens were evaluated for HPV-16and HPV-18with using the in situ hybridization catalyzed signal amplification method for biotinylated probes. The5-year overall survival (OS) and progression free survival (PFS) were calculated in relation to HPV status and the Cox proportional hazards model was used to determine the hazard ratio (HR) of variables in univariate and multivariate analysis.Results:One hundred and five patients (84men and21women) were included in this study. The median age of the patients was60(range,42-78) years at the date of surgery. Follow up was complete. HPV was detected in27.6%(29) of the105patients with ESCC, and all positive cases were HPV-16. None were positive for HPV-18DNA. There were no significant differences between the two groups with respect to gender, age, pT status, pN status, TNM stage (AJCC), differentiation grade, adjuvant therapy, smoking, and alcohol consumption and tumor location. The5-year OS rate and PFS rate were65.9%and61.8%, respectively, in the HPV-positive group and43.3%,36.8%, respectively, in the HPV-negative group. The difference of survival curves between the two groups remained statistically significant. In the Cox proportional hazards model analysis of5-year survival, the HR of HPV-positive/negative was0.31(95%CI:0.14-0.68; P=0.004) and0.37(95%CI:0.16-0.82; P=0.01), respectively. In the Cox proportional hazards model analysis of5-year PFS, the HR for HPV-positive/negative was0.33(95%CI:0.16-0.67; P=0.002) and0.38(95%CI:0.18-0.77; P=0.008), respectively. HPV infected patients had better5-year rates of OS and PFS then HPV-negative group (P=0.004and P=0.002by the Log-rank test, respectively). After adjustment for pT status, pN status, TNM stage, and smoking, tumor HPV status was also statistically significantly associated with survival among patients with ESCC.Conclusion:HPV infection may be one of many factors contributing to the development of ESCC and tumor HPV status is an independent prognostic factor for survival among patients with ESCC.Part2Study of correlation between p16INK4a and HPV infection in ESCCObjective:HPV-positive tumors are characterized by high expression of pl6and p16is widely considered a surrogate marker for HPV infection in the context of squamous cell carcinoma. This section is intended to explore the feasibility of p16immunohistochemical staining as an alternative molecular marker of HPV infection in ESCC.Method:A total of105patients who underwent esophagectomy for ESCC were included in this study. All specimens were tested for p16expression by immunohistochemistry. All slides were reviewed by a pathologist specializing in gastrointestinal pathology. Kappa values were calculated with the use of Cohen Kappa test. Using p16expression as a stratification factor, we calculated5-year OS and PFS through the Kaplan-Meier method and Log-rank test. Results:One hundred and five patients (84men and21women) were included in this study. The median age of the patients was60(range,42-78) years at the date of surgery. The median follow-up time was42.4(4-61) months of patients. Follow up was complete. P16was detected in37.1%(39) of the105patients with ESCC. There were no significant differences between p16-positive group and-negative with respect to gender, age, pT status, pN status, TNM stage (AJCC), differentiation grade, adjuvant therapy, smoking, and alcohol consumption and tumor location. P16was detected in86.2%(25) of the29patients who were HPV-positive, and p16was detected in18.4%(14) of the76patients who were HPV-negative (P<0.001). Cohen’s kappa coefficient revealed an agreement in two researchers (kappa=0.61). The5-year OS rate and PFS rate were64.1%and58.7%, respectively, in the p16-positive group and45.5%,37.9%, respectively, in the p16-negative group. The difference of survival curves between the two groups remained statistically significant. P16-positive patients had better5-year rates of OS and PFS then p16-negative group (P=0.02and P=0.007by the Log-rank test, respectively). Using p16expression as a stratification factor, we found differences in OS and PFS that were consistent with those based on HPV status.Conclusion:HPV-positive ESCC tumors were characterized by overexpression of p16protein, p16immunohistochemistry is a very good surrogate marker of HPV infection for ESCC.Conclusion of the study:HPV infection may play an important role in the development of ESCC. P16protein immunohistochemistry is a surrogate marker of HPV infection for ESCC. HPV status in tumor is an independent prognostic factor for survival among patients with ESCC. HPV infection may become a new therapeutic target in ESCC.更多还原