河南食管癌高发区单发食管癌、贲门癌及同一个体食管/贲门双源癌人乳头瘤病毒检测和p53、p16、p21~(WAF1)及MDM2蛋白变化研究
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摘要
1.研究背景与目的
食管癌(Esophageal carcinoma, EC)是最常见的六大恶性肿瘤之一,河南省林州地区(以前林县)是世界上发病率最高的地区,组织类型以食管鳞癌(Esophageal squamous cell carcinoma, SCC)为主(95%)。男、女发病率分别高达161/10万、103/10万,局部地区可高达500/10万,且预后极差,中晚期患者5年存活率仅为10%,早期患者5年存活率可达90%。目前为止,高发的原因尚未清楚。以往的研究认为：高发区主要分布在发展中国家,常常因为饮食结构单一,缺乏足够的绿色蔬菜和新鲜水果的摄入,导致维生素、抗氧化剂和微量元素缺乏,再合并摄入致癌剂如亚硝胺或含真菌的霉变食物时,就容易导致食管癌的发生。食管粘膜是外界环境因素频繁接触的部位,是一个外来物进入体内的重要通道。这些外来物中,包括一些病原微生物,化学致癌物,食品添加剂和环境污染物等。多年的流行病学调查及实验研究表明某些微生物与消化道肿瘤的发生关系密切。
人乳头瘤病毒(Human papillomavirus, HPV)作为一个重要的肿瘤相关病毒,其在宫颈癌中的作用已得到公认。至于它在食管癌中的作用,自1982年Syrjanen首次报道以来,HPV感染与食管癌发病之间的关系逐渐引起人们的重视,相关的报道越来越多。来自不同国家及地区报告食管癌中人乳头瘤病毒的检出率相差很大,甚至相悖,最低及最高HPV检测率均来自河南林州食管癌高发区,除此之外,国外的南非和印度HPV感染率报告也较高。而在欧洲与北美的一些发达国家,HPV在食管癌中的检出率较低。以往研究所用的检测方法主要有针对HPV表达蛋白改变的免疫组化(IHC),针对HPV mRNA的原位杂交(ISH)及多聚酶链式反应(PCR),或者针对抗体的血清学检测。
对这些不同的检测结果,多数人倾向认为可能与地域不同、检测方法不同以及取材部位不同有关,但对采用相同的检测技术,若标本的来源形式不同,检测结果是否有差别却很少有报道。
通常所说的贲门是指胃食管交界线以下2cm的范围,解剖结构与生理功能上与食管有很多相似之处。在食管癌高发区一个显著的流行病学特征是贲门腺癌(Gastric cardia adenocarcinoma，GCA)发病率也高,而远端胃癌的发病率较低,这种现象也见于其它食管癌高发区。
本研究旨在通过应用聚合酶链式反应(polymerase chain reaction, PCR),对不同组织来源形式(新鲜组织与石蜡固定组织)的单发食管癌、贲门癌标本进行检测,同时以高发区胃镜食管粘膜活检组织作对照,分析HPV检测差异产生的可能原因。在此基础上,借助本地独特的食管/贲门双源癌这一病例优势,进一步探讨HPV感染与p53、p16、p21WAF1及MDM2蛋白变化之间的关系。加深对食管贲门癌变机理,食管责门癌高易感性分子机制,肿瘤多中心起源理论的了解,特别是为进一步回答该地区食管鳞癌和贲门腺癌并存高发是否具有相似的致癌危险因素和癌变分子基础等方面提供一些理论依据。
2.材料与方法
2.1研究对象
食管癌患者：共44例,来自河南省林州市食管癌医院及林州市中心医院(男26例,平均年龄59±7；女18例,平均年龄56±9)。术后病理检查均为食管鳞癌。
贲门癌患者：共18例,来自河南省林州市食管癌医院及林州市中心医院(男13例,平均年龄60±8；女5例,平均年龄61±10)。术后病理检查均为贲门腺癌。
石蜡固定组织：与新鲜食管癌组织对应的石蜡固定组织30例(男18例,平均年龄57±8；女12例,平均年龄57±10)。病理检查均为食管鳞癌,高分化鳞癌3例,中分化鳞癌23例,低分化鳞癌4例；合并有淋巴结转移20例,无淋巴结转移10例。
食管/贲门双源癌组织：共17例,来自河南省林州市中心医院,姚村食管癌医院(男12例,平均年龄59±7；女5例,平均年龄58±9),所有患者术前均未接受放疗和化疗。
健康对照组：共18例,来自食管癌高发区无症状普查居民(男9例,平均年龄50±11；女9例,平均年龄48±10)。均经胃镜检查和活检病理证实为无食管炎及食管各级癌前疾病。
2.2标本的收集和处理
各种手术切除标本均一分为二,一半固定；一半冰冻(-80℃),常规组织学处理,光镜观察,备用。
2.3方法
2.3.1新鲜组织DNA的提取：
新鲜组织DNA采用QIAamp DNA mini kit试剂盒抽提,取25mg组织研钵碾碎。加180ul ATL和20μl蛋白酶K,于1.5ml离心管56℃温浴1h,取离心管,加200μl AL,振荡,混匀。70℃水浴10min,加200μl乙醇,离心,8000rpm/min, 10min;上清液移入柱子,8000rpm/min, 1min;弃去洗脱液,分别加500μl AW1及AW2,8000rpm/min, 1min;弃去洗脱液,加AE 80μl8000rpm/min。收集DNA液,-20℃保存。
2.3.2石蜡固定组织DNA的提取：
切取10μm白片10-15张,56℃烘烤1h,脱蜡及梯度酒精至水,刮下组织收入EP管中,加入细胞裂解液600μl,蛋白酶K 80μl,55℃水浴24h。加600μlTris饱和酚,14000 rpm x 5 min;上清液转入新EP管中,加600μl饱和酚,14000rpm x 5 min;上清液转入EP管中加入500μl氯仿,14000 rpm×5 min；上清液转入EP管中,加入1000μl异丙醇,14000 rpm×5min,去上清,吸干试管；加入70%乙醇600μl,12000 rpm x 3 min,去上清,留沉淀；加去离子水80μl,分装后放入-20℃保存。
2.3.3免疫组织化学：
采用卵白素-生物素-辣根过氧化氢酶复合物(ABC)方法检测p53、p16、p21WAF1、MDM2蛋白的表达。用本实验室以往阳性切片做阳性对照,用磷酸盐缓冲液代替一抗做阴性对照。
免疫组化蛋白阳性细胞主要表现为细胞核呈黄色至棕黄色,其阳性标准,高倍镜下(×40)选取五个视野,出现3个或3个以上的细胞核、细胞浆和/或胞膜呈黄色、棕黄色或棕褐色颗粒样着色,即为免疫阳性反应。MDM2免疫组化蛋白阳性细胞主要表现为胞核棕黄色颗粒状、均质染色,个别细胞胞浆浅黄色着色。其阳性标准,根据阳性细胞分布和染色强度判定。高倍镜下(×40),无阳性细胞为0分,阳性细胞≤1/3为1分,1/3～2/3为2分,≥2/3为3分。染色强度按切片中细胞着色有无及深浅记分：细胞无着色0分；中度染色1分；染色强2分(高倍镜下能清楚的分辨)。将2分值相加,0=(-)；2=(±)；3=(+)；4=(++);5=(+++)。
2.3.4模板DNA质量鉴定
(1)在核酸/蛋白分析仪上测OD(260/280)值,进行实验样品DNA的OD值在1.6～2.0之间。
(2) 1%琼脂糖凝胶电泳,进一步确定所提DNA的质量。
(3)运用beta-actin进行内参扩增,内参扩增阴性的样品将会被排除,并排除PCR反应体系中抑制因子的影响。
2.3.5 HPV16E6序列PCR扩增
设计E6特异性引物,进行PCR扩增,反应总体积为50ul,其中含10×xbuffer 5.0μ, Mgcl2(25mmol/L) 5.0μl, dNTP(2.5mmol/L) 4.0μl上下游引物各1.0μl,Ex-Taq 0.12U, DNA template 1.0μl,加ddH20至50μl；循环包括95℃预变性5min,5个循环：95℃变性1min,55℃退火1min,72℃延伸1min,接着再40个循环：95℃变性20s,55℃退火30s,72℃延伸30s,最后72℃终延伸5min。经电泳PCR阳性产物为120bp条带。
2.3.6统计分析
采用SPSS13.0统计软件处理,采用x2检验,Fisher's Exact Test检验,Pearson correction相关分析等统计学方法进行分析,取α=0.05。
3结果
3.1 44例新鲜SCC组织HPV16 E6扩增结果
beta-actin内参扩增阳性的44例标本,针对HPV 16 E6特异性引物进行PCR检测结果表明,HPV在SCC患者新鲜组织中的检出率为84.1%(37/44)。高危型HPV16感染与患者年龄、性别、分化程度、浸润程度、淋巴结转移、肿瘤分级无相关性(P>0.05)。
3.2 18例新鲜GCA组织HPV16 E6扩增结果
beta-actin内参扩增阳性的18例标本,针对HPV16 E6特异性引物进行PCR检测结果表明,HPV在GCA患者新鲜组织中的检出率为44.4%(8/18)。高危型HPV16感染与患者年龄、性别、分化程度、浸润程度、淋巴结转移、肿瘤分级无相关性(P>0.05)。
3.3 18例胃镜食管粘膜活检组织HPV16 E6扩增结果
beta-actin内参扩增阳性的18例标本,针对HPV16 E6特异性引物进行PCR检测结果表明,HPV在食管癌高发区正常人胃镜食管粘膜活检组织中的检出率为33.3%(6/18)。
3.4与新鲜SCC组织配对的30例石蜡固定组织HPV16 E6扩增结果
30例配对完整的SCC新鲜组织与石蜡固定组织标本,新鲜组织DNA经PCR扩增后,HPV16 E6有27例出现阳性条带,3例为阴性条带；相对应的石蜡组织中3例阴性均为阴性条带,而27例阳性条带中仅16例出现阳性,其余11例为阴性,HPV在SCC患者固定组织中的检出率为53.3%(16/30)。经配对检验p<0.01,在排除方法本身应有局限性外,石蜡固定组织明显低于新鲜组织。
3.5 17例食管/贲门双源癌组织标本HPV16 E6扩增结果
beta-actin内参扩增,食管及贲门两者均阳性的标本17对,针对HPV16 E6特异性引物进行PCR检测结果表明,HPV在SCC组织中的检出率为47.1%(8/17),在GCA组织中的检出率为29.4%(5/17)。两者相比无显著性差异(p>0.05)。
3.6 17例食管/贲门双源癌免疫组化结果
3.6.1 p53免疫组化结果
SCC和GCA组织均出现不同程度的p53免疫阳性反应。17例双源癌,p53在SCC和GCA组织中阳性表达率均为64.7%(11/17),p53在SCC和GCA中阳性表达无显著性差异(p>0.05)。13例(76.5%)患者同时出现SCC和GCA组织一致性改变,其中9例(52.9%)患者SCC和GCA组织呈p53免疫一致阳性反应,4例(23.5%)同时出现p53的免疫阴性反应。统计学分析认为p53在SCC和GCA中一致性阳性表达有相关性(p<0.05),结合Pearson列联系数P=0.470,可认为p53在SCC和GCA中蛋白改变结果一致。
3.6.2 p16免疫组化结果
SCC和GCA组织均出现不同程度的p16免疫阳性反应。17例双源癌,SCC中p16阳性率为41.2%(7/17),GCA阳性占58.8%(10/17)。p16蛋白在12例患者同时出现SCC和GCA组织一致性改变(70.6%),其中6例患者SCC和GCA组织呈p16免疫阳性反应(35.3%),6例同时出现p16的免疫阴性反应(35.3%)。p16在SCC和GCA中阳性表达无显著性差异(p>0.05)。
3.6.3 p21WAF1免疫组化结果
SCC和GCA组织均出现不同程度的p21WAF1免疫阳性反应。17例双源癌,p21WAF1阳性率在SCC和GCA中均为41.2%(7/17),其中14例患者同时出现SCC和GCA组织一致性改变(82.4%),6例患者呈p21WAF1免疫阳性反应(35.3%),8例同时出现p21WAF1的免疫阴性反应(47.1%)。统计学分析认为p21WAF1在SCC和GCA中阳性表达有相关性,结合Pearson列联系数P=0.591,可认为p21WAF1在SCC和GCA中蛋白改变结果一致,p21WAF1在SCC和GCA中阳性表达差异有显著性(p<0.05)。
3.6.4 MDM2免疫组化结果
17例双源癌,MDM2蛋白在SCC和GCA组织中阳性率分别为58.8%(10/17),70.6%(12/17),MDM2在SCC和GCA中阳性表达无显著性差异(p>0.05)。15例(88.2%)患者同时出现MDM2蛋白一致性改变,其中10例(58.9%)患者SCC和GCA组织MDM2呈免疫阳性反应,5例(29.4%)同时出现MDM2的免疫阴性反应。
3.7 17例双源癌组织中HPV与p21WAF1、MDM2免疫组化结果对比分析
3.7.1 (?)17例食管/贲门双源癌中HPV与p53免疫组化结果对比分析
经Fisher's Exact Test检验,HPV感染和p53蛋白改变在SCC及GCA中P值分别为0.335及1.0,均大于0.05,说明HPV感染在食管及贲门癌中与p53蛋白改变结果一致,差别无统计学意义。
3.7.2 17例食管/贲门双源癌中HPV与p16免疫组化结果对比分析
经Fisher's Exact Test检验,p16蛋白改变和HPV感染在SCC及GCA中p值分别为0.335及0.338,均大于0.05,说明HPV感染在食管及贲门癌中与p16蛋白改变结果相同。
3.7.3 17例食管/贲门双源癌中HPV与p21WAF1免疫组化结果对比分析
经Fisher's Exact Test检验,HPV感染和p21WAF1改变在SCC及GCA中p值均为1.0,大于0.05,说明HPV感染在食管及贲门癌中与p21WAF1蛋白改变结果一致。
3.7.4 17例食管/贲门双源癌组织中HPV与MDM2免疫组化结果对比分析
经Fisher's Exact Test检验,MDM2蛋白改变和HPV感染在SCC及GCA中p值分别为0.637及1.0,均大于0.05,说明HPV感染在食管及贲门癌中与MDM2蛋白改变结果相同,差别无统计学意义。
4.结论
4.1高发区食管癌患者中HPV感染率明显高于健康对照组,提示HPV感染可能是该区食管癌高发的病因之一。但HPV感染与性别、年龄、肿瘤浸润深度、淋巴结转移、临床分期之间无关,是相互独立的。
4.2食管癌高发区贲门腺癌HPV的感染率也较高,提示HPV感染可能也参与该区贲门癌的发生过程。
4.3在高发区胃镜普查的食管粘膜上皮中,也可检出HPV病毒的感染(33.3%),提示HPV的感染先于癌变的进展,在高发区可能是一个促癌因素。
4.430例配对完整的食管癌新鲜组织与石蜡固定组织标本,采用相同方法扩增,HPV16 E6在两种组织中的检出率为分别为84.1%及53.3%。经配对检验P<0.01,在排除方法本身应有局限性外,石蜡固定组织明显低于新鲜组织。这可能与组织在固定及切取过程中,DNA完整性破坏,形成一些小的片段,以至于超出方法本身所具有的检测灵敏度有关。
4.5对食管/贲门双源癌患者(同一个体),HPV在SCC中的检出率为47.1%(8/17),在GCA中的检出率为29.4%(5/17)。两者相比无显著性差异(p>0.05),进一步提示在高发区HPV可能共同参与食管及贲门的癌变过程,两者具有相似的致癌危险因素。
4.6对食管/贲门双源癌患者,HPV感染与p53、p16、p21WAF1、MDM2蛋白改变相关,其中p53、p21WAF1、MDM2蛋白改变与贲门癌更相关,提示在癌变的演进过程中,两者可能具有不同的分子基础。
1. BACKGROUND AND OBJECTIVE
Esophageal cancer (EC) is one of the six most common malignant diseases worldwide. Linzhou of Henan province, Northern China, has been recognized as the highest incidence area for EC in the world, and the predominant histologic type in this area is squamous cell carcinoma (SCC), the incidence rates are around 161/100,000 for men and 103/100,000 for women. However, in distinct areas the incidence rates are remarkably higher, with up to 500 fold variations between low and high risk areas. EC in late stage has a very poor prognosis with a five year survival rate of less than 10%. However, the 5-year survival rate for EC in the early stage could as high as 90%. Up to now, the reasons for these major regional variations in the incidence of this disease are poorly understood. Epidemiological and experimental data suggest that some chemicals, nutritional deficiencies, physical factors, and infectious agents are associated with the development of this malignancy. An aetiological role of certain microorganisms has been implicated (direct carcinogens or promoters).
Human papillomavirus (HPV) as one kind of important tumor-related virus has been firmly recognized in cervical cancer. But its oncogenic role in other tumors is still disputed. As to its role in ESCC, it was firstly suggested by Syrjanen 20 years ago, when he reported an involvement of HPV in the development of both benign and malignant SCC of the esophagus, since then, a substantial amount of literatures have accumulated on this subject. But the HPV infection rate in EC varied from 0% to 100 %. There seems to be a common denominator for the low and high detection rates: namely, the geographical distribution of the material.
For different detection rates, the possible reasons:①the cases came from different areas where HPV prevalence was different.②many different HPV detection methods have been used. Different methods have different sensitivity and accuracy. Of them, PCR is more sensitive than others, but different primers have different sensitivity.③the samples taken from different parts, of esophagus. HPV only reproduce in those highly differentiated epidermis cells. The number of copies varies widely in different degree of differentiation, which may impact on the results. Linzhou area is one of the highest incidence areas of esophageal cancer in China, even in the world. In recent years, some studies have been performed on the prevalence of HPV in the tissues of esophageal cancer in Linzhou area, but the conclusions were not consistent. Therefore, it is still questionable whether HPV is one of the major causes of esophageal cancer in this high-risk area.
In epidemiology, gastric cardia adenocarcinoma (GCA) in China is characterized by its striking geographic distraction, Linzhou in Henan Province has been well documented as the highest incidence area for GCA. The other phenomenon in Linzhou is that the primary SCC and GCA could occur together on the same patient, which has been named as concurrent carcinoma (CC) of the esophagus and gastric cardia by us.
In order to explore the prevalence of HPV and alternation of p53, p16, p21WAF1, MDM2 in carcinoma tissues of local residents in Linzhou area and to evaluate their possible roles and interaction in the carcinogenesis, here we present the following experiments. Some useful findings may be provided scientific basis to prevent esophageal cancer.
2. MATERIALS AND METHODS
2.1 Subjects
Esophageal cancer:A total of 44 surgically resected human EC specimens were recruited from Linzhou City Center Hospital and Yaocun Esophageal Cancer Hospital in Linzhou, Henan, the highest incidence area for both EC and GCA, including 26 males with an average age of 59±7 years old and 18 females with an average age of 56±9 years old.
Gastric cardia:All the 18 surgically resected human GCA specimens were collected from the same hospitals as above. Of the 18 cases,13 were males with an average age of 60±8 years old and 5 females with an average age of 61±10 years old.
Biospy tissue:All the 18 endoscopic biopsy esophageal epithelium as normal control were collected from high-incidence regions. Of the 18 cases,9 were males with an average age of 48±10 years old and 9 females with an average age of 50±11 years old.
Paraffin-embedded tissue:All the 30 surgically resected human EC specimens were collected from the same hospitals as above. Of the 30 cases,18 were males with an average age of 57±8 years old and 12 females with an average age of 57±10 years old.
CC patients:All the 17 cases patients with CC in this study were from the same hospitals as above. Of the patients,12 were males and 5 were females, with a mean age of 59±7 years in male and 58±9 years in female. All the CC patients were conformed to primary SCC and GCA from the same patient by histopathology.
None of the patients had received chemotherapy and/or radiotherapy before operation. All the cases were confirmed as GCA or SCC respectively by histopathology. TNM staging was undertaken based on the criteria by AJCC (2002, AJCC).
2.2 Tissue collection and processing
Within 2 hours after surgical excision, the specimens were cut into two-halves longitudinally. Half of the specimen was fixed with 85% ethanol, paraffin-embedded, and sectioned at 5μm for histopathological evaluation and immunohistochemical analysis. The other part of the specimens was stored in the liquid nitrogen first and then transferred in a-80℃freezer for DNA extraction.
2.3 Methods
2.3.1 DNA extraction for frozen tissue
DNA extraction from the SCC and GCA pathology sample was performed with QIAamp DNA mini kit, QIAGEN company DNA mini kitTM (51306).
2.3.2 DNA extraction for paraffin-embedded tissue
Briefly,10-15 slides were deparaffinized in xylene and graded alcohol, then the lysis buffer (300 mmol/1 NaCl; 50 mmol/1 Tris.hCl pH 8.0; 0.2% SDS) was added into the tube with proteinase K (0.5 mg/ml), and the solution was incubated at 55℃overnight until it became clear. Then DNA was extracted using phenol/chloroform, precipitated with cold alcohol, and dissolved in ion-free water and stored at-20℃
2.3.3 Immunohistochemical analysis
Avidin-biotin-peroxidase complex (ABC) method was used to analyze the expression of p53, p16, p21WAF1 and MDM2. The classification of immunoreactivity was based on the immunostaining intensity (score as 0,1,2,3) and cell proportion of positive immunostaining (score as 0,1,2,3,4).
2.3.4 Identification for the purity and integrity of DNA samples
The DNA concentration was determined from its optical density. Quality of the extracted DNA was tested by PCR with beta-actin primer:5'-TCA CCC ACA CTG TGC CCA TC-3'and 5'-GAA CCG CTC ATT GCC AAT GG-3'.
2.3.5 HPV16 E6 amplification
The usable DNA went through PCR amplification using primer:5'-TCA AAA GCC ACT GTG TCC TG-3'and 5'-CGT GTT CTT GAT GAT CTG CA-3'targeting HPV-16 E6 gene under conditions at 95℃denaturing for 5 min; at 95℃denaturing for 1 min,55℃annealing for 1 min, and at 72℃prolonging for 1 min with 5 cycles; then at 95℃denaturing for 20s,55℃annealing for 30s, and at 72℃prolonging for 30s with 40 cycles; at 72℃prolonging for 5 min. The PCR product was about 120 bp. The plasmid containing full of length of HPV-16 genome as template was the positive control, and the water as template was the negative control.
2.3.6 Statistics
Chi-squared test and Kappa test were performed to compare the difference between groups.
3. RESULTS
HPV16 E6 amplification for 44 EC frozen tissues
All the forty-four extracted DNA samples showed good quality of DNA after PCR with beta-actin primer. After PCR amplification using HPV-16 E6 specific primer, HPV infection was found in tumor patients for 84.1% (37/44) in Linzhou. There was no association between HPV16 infection and gender, age, differentiation, lymph node metastasis and tumor staging.
HPV16 E6 amplification for 18 GCA frozen tissues
All the eighteen extracted frozen gastric cardia cancer samples showed good quality of DNA after PCR with beta-actin primer. PCR amplification using HPV-16 E6 specific primer, HPV infection was found in tumor patients for 44.4%(8/18) in Linzhou, risk incidence area of esophageal cancer. No association between HPV 16 infection and gender, age, differentiation, lymph node metastasis and tumor staging.
HPV16 E6 amplification for 18 biospy esophageal epithelium
Using the same method, HPV infection rate was found in endoscopic biopsy esophageal epithelium as normal control was collected from high-incidence regions for33.3%(6/18).
HPV16 E6 amplification for 30 paraffin-embedded
Using the same method, HPV infection rate was found in frozen sample of esophagus for 84.1% and 53.3%(16/30) from paraffin-embedded tissue, respectively. But comparing the positive rate of frozen and paraffin-embedded samples, the difference of HPV infection in DNA level was significant (P< 0.05).
HPV 16 E6 amplification for 17 CC
All the extracted DNA samples showed good quality of DNA after PCR with beta-actin primer. After PCR amplification using HPV-16 E6 specific primer, HPV infection was found in tumor patients from both tissue, with an infection rate of 47.1% (8/17) in SCC and 29.4% in GCA. There have no significant differences among them by statistics (P> 0.05).
Immunohistochemical staining for 17 CC
In CC patients, the positive immunohistochemical staining for p53, p16, p21WAF1 and MDM2 was observed both in SCC and GCA with different degrees. In SCC, the overexpression rates of p53 was the highest with 64.7%(11/17), followed by MDM2, p16 and p21WAF1 with 58.8%(10/17),41.2%(7/17) and 41.2%(7/17), respectively. In GCA, the overexpression rates of MDM2, p53, p16 and p21WAF1 were similar, with 70.6%(12/17),64.7%(11/17),58.8%(10/17) and 47.1%(8/17), respectively. Frequent immunostaining pattern for MDM2 and p53 was "Diffuse" and "Scatter". In p16, and p21WAF1, "Scatter" immunostaining pattern was predominant. High coincidence rates for MDM2, p53, p16 and p21WAF1 positive staining was observed in SCC and GCA from the same patients, and were 70.6%(12/17),64.7%(11/17),58.8%(10/17) and 47.1%(8/17), respectively.
Comparison for HPV infection and p53, p16, p21WAF1, MDM2 immunohistoche-mical staining in 17 CC
In CC patients, correction was analyed both in SCC and GCA with HPV infection and immunohistochemical staining for p53, p16, p21WAF1 and MDM2. In GCA, the correction of HPV infection and p53, p21WAF1, MDM2 were the highest for 1.0, followed by p16 for 0.338, respectively. In SCC, the correction of HPV infection and p53, p16 and p21WAF1, MDM2 were similar, with 0.335,0.335,1.0 and 0.637, respectively.
4. CONCLUSIONS
4.1 The prevalence of HPV16 in the esophageal cancer patients was higher than that gastric cardia cancer and healthy control subjects, which indicate that high-risk HPV plays an important role in the development of esophageal cancer of risk-incidence areas. Statistical analyses show that the prevalence of HPV is not dependent on gender, age, differentiation, lymph node metastasis and tumor staging.
4.2 The incidence of gastric cardia adenocarcinoma is also very high in this area, higher detection rate of HPV in gastric cardia adenocarcinoma indicate that HPV may be an aetiological factor in carcinogenesis of GCA.
4.3 HPV DNA was amplified with a high detection rate in endoscopic biopsy healthy subjects in risk incidence area show HPV as a tumor promoters preceding carcinogenesis progression.
4.4 30 cases of pair integrity esophageal cancer sample, the fresh(frozen) and paraffin- embedded tissues amplified using the same approach, the two organizations HPV16 E6 in the detection rate was 84.1% and 53.3%, respectively. The matching test, (P<0.01); the limitations of method itself should be excluded, paraffin-embedded tissues was significantly lower than fresh tissue. This may be fixed and all organizations in the process, the DNA breakdown, the formation of some of the smaller fragments.
4.5 The CC patients (the same individual), and HPV in the SCC in the detection rate of 47.1%(8/17), the detection rate was 29.4%(5/17) in the GCA. When comparing the two no significant difference (P> 0.05). HPV infection may suggest that the high incidence area of common participation in the esophagus and gastric cardia cancer process, the two have similar carcinogenic risk factors.
4.6 For CC patients, HPV infection and p53, p16, p21WAF1, MDM2 protein changes associated, Which p53, p21WAF1, MDM2 protein changes associated with more GCA, Suggest that the evolution of the process of carcinogenesis may have different molecular basis.
食管癌(Esophageal carcinoma, EC)是最常见的六大恶性肿瘤之一,河南省林州地区(以前林县)是世界上发病率最高的地区,组织类型以食管鳞癌(Esophageal squamous cell carcinoma, SCC)为主(95%)。男、女发病率分别高达161/10万、103/10万,局部地区可高达500/10万,且预后极差,中晚期患者5年存活率仅为10%,早期患者5年存活率可达90%。目前为止,高发的原因尚未清楚。以往的研究认为：高发区主要分布在发展中国家,常常因为饮食结构单一,缺乏足够的绿色蔬菜和新鲜水果的摄入,导致维生素、抗氧化剂和微量元素缺乏,再合并摄入致癌剂如亚硝胺或含真菌的霉变食物时,就容易导致食管癌的发生。食管粘膜是外界环境因素频繁接触的部位,是一个外来物进入体内的重要通道。这些外来物中,包括一些病原微生物,化学致癌物,食品添加剂和环境污染物等。多年的流行病学调查及实验研究表明某些微生物与消化道肿瘤的发生关系密切。
人乳头瘤病毒(Human papillomavirus, HPV)作为一个重要的肿瘤相关病毒,其在宫颈癌中的作用已得到公认。至于它在食管癌中的作用,自1982年Syrjanen首次报道以来,HPV感染与食管癌发病之间的关系逐渐引起人们的重视,相关的报道越来越多。来自不同国家及地区报告食管癌中人乳头瘤病毒的检出率相差很大,甚至相悖,最低及最高HPV检测率均来自河南林州食管癌高发区,除此之外,国外的南非和印度HPV感染率报告也较高。而在欧洲与北美的一些发达国家,HPV在食管癌中的检出率较低。以往研究所用的检测方法主要有针对HPV表达蛋白改变的免疫组化(IHC),针对HPV mRNA的原位杂交(ISH)及多聚酶链式反应(PCR),或者针对抗体的血清学检测。
对这些不同的检测结果,多数人倾向认为可能与地域不同、检测方法不同以及取材部位不同有关,但对采用相同的检测技术,若标本的来源形式不同,检测结果是否有差别却很少有报道。
通常所说的贲门是指胃食管交界线以下2cm的范围,解剖结构与生理功能上与食管有很多相似之处。在食管癌高发区一个显著的流行病学特征是贲门腺癌(Gastric cardia adenocarcinoma，GCA)发病率也高,而远端胃癌的发病率较低,这种现象也见于其它食管癌高发区。
本研究旨在通过应用聚合酶链式反应(polymerase chain reaction, PCR),对不同组织来源形式(新鲜组织与石蜡固定组织)的单发食管癌、贲门癌标本进行检测,同时以高发区胃镜食管粘膜活检组织作对照,分析HPV检测差异产生的可能原因。在此基础上,借助本地独特的食管/贲门双源癌这一病例优势,进一步探讨HPV感染与p53、p16、p21WAF1及MDM2蛋白变化之间的关系。加深对食管贲门癌变机理,食管责门癌高易感性分子机制,肿瘤多中心起源理论的了解,特别是为进一步回答该地区食管鳞癌和贲门腺癌并存高发是否具有相似的致癌危险因素和癌变分子基础等方面提供一些理论依据。
2.材料与方法
2.1研究对象
食管癌患者：共44例,来自河南省林州市食管癌医院及林州市中心医院(男26例,平均年龄59±7；女18例,平均年龄56±9)。术后病理检查均为食管鳞癌。
贲门癌患者：共18例,来自河南省林州市食管癌医院及林州市中心医院(男13例,平均年龄60±8；女5例,平均年龄61±10)。术后病理检查均为贲门腺癌。
石蜡固定组织：与新鲜食管癌组织对应的石蜡固定组织30例(男18例,平均年龄57±8；女12例,平均年龄57±10)。病理检查均为食管鳞癌,高分化鳞癌3例,中分化鳞癌23例,低分化鳞癌4例；合并有淋巴结转移20例,无淋巴结转移10例。
食管/贲门双源癌组织：共17例,来自河南省林州市中心医院,姚村食管癌医院(男12例,平均年龄59±7；女5例,平均年龄58±9),所有患者术前均未接受放疗和化疗。
健康对照组：共18例,来自食管癌高发区无症状普查居民(男9例,平均年龄50±11；女9例,平均年龄48±10)。均经胃镜检查和活检病理证实为无食管炎及食管各级癌前疾病。
2.2标本的收集和处理
各种手术切除标本均一分为二,一半固定；一半冰冻(-80℃),常规组织学处理,光镜观察,备用。
2.3方法
2.3.1新鲜组织DNA的提取：
新鲜组织DNA采用QIAamp DNA mini kit试剂盒抽提,取25mg组织研钵碾碎。加180ul ATL和20μl蛋白酶K,于1.5ml离心管56℃温浴1h,取离心管,加200μl AL,振荡,混匀。70℃水浴10min,加200μl乙醇,离心,8000rpm/min, 10min;上清液移入柱子,8000rpm/min, 1min;弃去洗脱液,分别加500μl AW1及AW2,8000rpm/min, 1min;弃去洗脱液,加AE 80μl8000rpm/min。收集DNA液,-20℃保存。
2.3.2石蜡固定组织DNA的提取：
切取10μm白片10-15张,56℃烘烤1h,脱蜡及梯度酒精至水,刮下组织收入EP管中,加入细胞裂解液600μl,蛋白酶K 80μl,55℃水浴24h。加600μlTris饱和酚,14000 rpm x 5 min;上清液转入新EP管中,加600μl饱和酚,14000rpm x 5 min;上清液转入EP管中加入500μl氯仿,14000 rpm×5 min；上清液转入EP管中,加入1000μl异丙醇,14000 rpm×5min,去上清,吸干试管；加入70%乙醇600μl,12000 rpm x 3 min,去上清,留沉淀；加去离子水80μl,分装后放入-20℃保存。
2.3.3免疫组织化学：
采用卵白素-生物素-辣根过氧化氢酶复合物(ABC)方法检测p53、p16、p21WAF1、MDM2蛋白的表达。用本实验室以往阳性切片做阳性对照,用磷酸盐缓冲液代替一抗做阴性对照。
免疫组化蛋白阳性细胞主要表现为细胞核呈黄色至棕黄色,其阳性标准,高倍镜下(×40)选取五个视野,出现3个或3个以上的细胞核、细胞浆和/或胞膜呈黄色、棕黄色或棕褐色颗粒样着色,即为免疫阳性反应。MDM2免疫组化蛋白阳性细胞主要表现为胞核棕黄色颗粒状、均质染色,个别细胞胞浆浅黄色着色。其阳性标准,根据阳性细胞分布和染色强度判定。高倍镜下(×40),无阳性细胞为0分,阳性细胞≤1/3为1分,1/3～2/3为2分,≥2/3为3分。染色强度按切片中细胞着色有无及深浅记分：细胞无着色0分；中度染色1分；染色强2分(高倍镜下能清楚的分辨)。将2分值相加,0=(-)；2=(±)；3=(+)；4=(++);5=(+++)。
2.3.4模板DNA质量鉴定
(1)在核酸/蛋白分析仪上测OD(260/280)值,进行实验样品DNA的OD值在1.6～2.0之间。
(2) 1%琼脂糖凝胶电泳,进一步确定所提DNA的质量。
(3)运用beta-actin进行内参扩增,内参扩增阴性的样品将会被排除,并排除PCR反应体系中抑制因子的影响。
2.3.5 HPV16E6序列PCR扩增
设计E6特异性引物,进行PCR扩增,反应总体积为50ul,其中含10×xbuffer 5.0μ, Mgcl2(25mmol/L) 5.0μl, dNTP(2.5mmol/L) 4.0μl上下游引物各1.0μl,Ex-Taq 0.12U, DNA template 1.0μl,加ddH20至50μl；循环包括95℃预变性5min,5个循环：95℃变性1min,55℃退火1min,72℃延伸1min,接着再40个循环：95℃变性20s,55℃退火30s,72℃延伸30s,最后72℃终延伸5min。经电泳PCR阳性产物为120bp条带。
2.3.6统计分析
采用SPSS13.0统计软件处理,采用x2检验,Fisher's Exact Test检验,Pearson correction相关分析等统计学方法进行分析,取α=0.05。
3结果
3.1 44例新鲜SCC组织HPV16 E6扩增结果
beta-actin内参扩增阳性的44例标本,针对HPV 16 E6特异性引物进行PCR检测结果表明,HPV在SCC患者新鲜组织中的检出率为84.1%(37/44)。高危型HPV16感染与患者年龄、性别、分化程度、浸润程度、淋巴结转移、肿瘤分级无相关性(P>0.05)。
3.2 18例新鲜GCA组织HPV16 E6扩增结果
beta-actin内参扩增阳性的18例标本,针对HPV16 E6特异性引物进行PCR检测结果表明,HPV在GCA患者新鲜组织中的检出率为44.4%(8/18)。高危型HPV16感染与患者年龄、性别、分化程度、浸润程度、淋巴结转移、肿瘤分级无相关性(P>0.05)。
3.3 18例胃镜食管粘膜活检组织HPV16 E6扩增结果
beta-actin内参扩增阳性的18例标本,针对HPV16 E6特异性引物进行PCR检测结果表明,HPV在食管癌高发区正常人胃镜食管粘膜活检组织中的检出率为33.3%(6/18)。
3.4与新鲜SCC组织配对的30例石蜡固定组织HPV16 E6扩增结果
30例配对完整的SCC新鲜组织与石蜡固定组织标本,新鲜组织DNA经PCR扩增后,HPV16 E6有27例出现阳性条带,3例为阴性条带；相对应的石蜡组织中3例阴性均为阴性条带,而27例阳性条带中仅16例出现阳性,其余11例为阴性,HPV在SCC患者固定组织中的检出率为53.3%(16/30)。经配对检验p<0.01,在排除方法本身应有局限性外,石蜡固定组织明显低于新鲜组织。
3.5 17例食管/贲门双源癌组织标本HPV16 E6扩增结果
beta-actin内参扩增,食管及贲门两者均阳性的标本17对,针对HPV16 E6特异性引物进行PCR检测结果表明,HPV在SCC组织中的检出率为47.1%(8/17),在GCA组织中的检出率为29.4%(5/17)。两者相比无显著性差异(p>0.05)。
3.6 17例食管/贲门双源癌免疫组化结果
3.6.1 p53免疫组化结果
SCC和GCA组织均出现不同程度的p53免疫阳性反应。17例双源癌,p53在SCC和GCA组织中阳性表达率均为64.7%(11/17),p53在SCC和GCA中阳性表达无显著性差异(p>0.05)。13例(76.5%)患者同时出现SCC和GCA组织一致性改变,其中9例(52.9%)患者SCC和GCA组织呈p53免疫一致阳性反应,4例(23.5%)同时出现p53的免疫阴性反应。统计学分析认为p53在SCC和GCA中一致性阳性表达有相关性(p<0.05),结合Pearson列联系数P=0.470,可认为p53在SCC和GCA中蛋白改变结果一致。
3.6.2 p16免疫组化结果
SCC和GCA组织均出现不同程度的p16免疫阳性反应。17例双源癌,SCC中p16阳性率为41.2%(7/17),GCA阳性占58.8%(10/17)。p16蛋白在12例患者同时出现SCC和GCA组织一致性改变(70.6%),其中6例患者SCC和GCA组织呈p16免疫阳性反应(35.3%),6例同时出现p16的免疫阴性反应(35.3%)。p16在SCC和GCA中阳性表达无显著性差异(p>0.05)。
3.6.3 p21WAF1免疫组化结果
SCC和GCA组织均出现不同程度的p21WAF1免疫阳性反应。17例双源癌,p21WAF1阳性率在SCC和GCA中均为41.2%(7/17),其中14例患者同时出现SCC和GCA组织一致性改变(82.4%),6例患者呈p21WAF1免疫阳性反应(35.3%),8例同时出现p21WAF1的免疫阴性反应(47.1%)。统计学分析认为p21WAF1在SCC和GCA中阳性表达有相关性,结合Pearson列联系数P=0.591,可认为p21WAF1在SCC和GCA中蛋白改变结果一致,p21WAF1在SCC和GCA中阳性表达差异有显著性(p<0.05)。
3.6.4 MDM2免疫组化结果
17例双源癌,MDM2蛋白在SCC和GCA组织中阳性率分别为58.8%(10/17),70.6%(12/17),MDM2在SCC和GCA中阳性表达无显著性差异(p>0.05)。15例(88.2%)患者同时出现MDM2蛋白一致性改变,其中10例(58.9%)患者SCC和GCA组织MDM2呈免疫阳性反应,5例(29.4%)同时出现MDM2的免疫阴性反应。
3.7 17例双源癌组织中HPV与p21WAF1、MDM2免疫组化结果对比分析
3.7.1 (?)17例食管/贲门双源癌中HPV与p53免疫组化结果对比分析
经Fisher's Exact Test检验,HPV感染和p53蛋白改变在SCC及GCA中P值分别为0.335及1.0,均大于0.05,说明HPV感染在食管及贲门癌中与p53蛋白改变结果一致,差别无统计学意义。
3.7.2 17例食管/贲门双源癌中HPV与p16免疫组化结果对比分析
经Fisher's Exact Test检验,p16蛋白改变和HPV感染在SCC及GCA中p值分别为0.335及0.338,均大于0.05,说明HPV感染在食管及贲门癌中与p16蛋白改变结果相同。
3.7.3 17例食管/贲门双源癌中HPV与p21WAF1免疫组化结果对比分析
经Fisher's Exact Test检验,HPV感染和p21WAF1改变在SCC及GCA中p值均为1.0,大于0.05,说明HPV感染在食管及贲门癌中与p21WAF1蛋白改变结果一致。
3.7.4 17例食管/贲门双源癌组织中HPV与MDM2免疫组化结果对比分析
经Fisher's Exact Test检验,MDM2蛋白改变和HPV感染在SCC及GCA中p值分别为0.637及1.0,均大于0.05,说明HPV感染在食管及贲门癌中与MDM2蛋白改变结果相同,差别无统计学意义。
4.结论
4.1高发区食管癌患者中HPV感染率明显高于健康对照组,提示HPV感染可能是该区食管癌高发的病因之一。但HPV感染与性别、年龄、肿瘤浸润深度、淋巴结转移、临床分期之间无关,是相互独立的。
4.2食管癌高发区贲门腺癌HPV的感染率也较高,提示HPV感染可能也参与该区贲门癌的发生过程。
4.3在高发区胃镜普查的食管粘膜上皮中,也可检出HPV病毒的感染(33.3%),提示HPV的感染先于癌变的进展,在高发区可能是一个促癌因素。
4.430例配对完整的食管癌新鲜组织与石蜡固定组织标本,采用相同方法扩增,HPV16 E6在两种组织中的检出率为分别为84.1%及53.3%。经配对检验P<0.01,在排除方法本身应有局限性外,石蜡固定组织明显低于新鲜组织。这可能与组织在固定及切取过程中,DNA完整性破坏,形成一些小的片段,以至于超出方法本身所具有的检测灵敏度有关。
4.5对食管/贲门双源癌患者(同一个体),HPV在SCC中的检出率为47.1%(8/17),在GCA中的检出率为29.4%(5/17)。两者相比无显著性差异(p>0.05),进一步提示在高发区HPV可能共同参与食管及贲门的癌变过程,两者具有相似的致癌危险因素。
4.6对食管/贲门双源癌患者,HPV感染与p53、p16、p21WAF1、MDM2蛋白改变相关,其中p53、p21WAF1、MDM2蛋白改变与贲门癌更相关,提示在癌变的演进过程中,两者可能具有不同的分子基础。
1. BACKGROUND AND OBJECTIVE
Esophageal cancer (EC) is one of the six most common malignant diseases worldwide. Linzhou of Henan province, Northern China, has been recognized as the highest incidence area for EC in the world, and the predominant histologic type in this area is squamous cell carcinoma (SCC), the incidence rates are around 161/100,000 for men and 103/100,000 for women. However, in distinct areas the incidence rates are remarkably higher, with up to 500 fold variations between low and high risk areas. EC in late stage has a very poor prognosis with a five year survival rate of less than 10%. However, the 5-year survival rate for EC in the early stage could as high as 90%. Up to now, the reasons for these major regional variations in the incidence of this disease are poorly understood. Epidemiological and experimental data suggest that some chemicals, nutritional deficiencies, physical factors, and infectious agents are associated with the development of this malignancy. An aetiological role of certain microorganisms has been implicated (direct carcinogens or promoters).
Human papillomavirus (HPV) as one kind of important tumor-related virus has been firmly recognized in cervical cancer. But its oncogenic role in other tumors is still disputed. As to its role in ESCC, it was firstly suggested by Syrjanen 20 years ago, when he reported an involvement of HPV in the development of both benign and malignant SCC of the esophagus, since then, a substantial amount of literatures have accumulated on this subject. But the HPV infection rate in EC varied from 0% to 100 %. There seems to be a common denominator for the low and high detection rates: namely, the geographical distribution of the material.
For different detection rates, the possible reasons:①the cases came from different areas where HPV prevalence was different.②many different HPV detection methods have been used. Different methods have different sensitivity and accuracy. Of them, PCR is more sensitive than others, but different primers have different sensitivity.③the samples taken from different parts, of esophagus. HPV only reproduce in those highly differentiated epidermis cells. The number of copies varies widely in different degree of differentiation, which may impact on the results. Linzhou area is one of the highest incidence areas of esophageal cancer in China, even in the world. In recent years, some studies have been performed on the prevalence of HPV in the tissues of esophageal cancer in Linzhou area, but the conclusions were not consistent. Therefore, it is still questionable whether HPV is one of the major causes of esophageal cancer in this high-risk area.
In epidemiology, gastric cardia adenocarcinoma (GCA) in China is characterized by its striking geographic distraction, Linzhou in Henan Province has been well documented as the highest incidence area for GCA. The other phenomenon in Linzhou is that the primary SCC and GCA could occur together on the same patient, which has been named as concurrent carcinoma (CC) of the esophagus and gastric cardia by us.
In order to explore the prevalence of HPV and alternation of p53, p16, p21WAF1, MDM2 in carcinoma tissues of local residents in Linzhou area and to evaluate their possible roles and interaction in the carcinogenesis, here we present the following experiments. Some useful findings may be provided scientific basis to prevent esophageal cancer.
2. MATERIALS AND METHODS
2.1 Subjects
Esophageal cancer:A total of 44 surgically resected human EC specimens were recruited from Linzhou City Center Hospital and Yaocun Esophageal Cancer Hospital in Linzhou, Henan, the highest incidence area for both EC and GCA, including 26 males with an average age of 59±7 years old and 18 females with an average age of 56±9 years old.
Gastric cardia:All the 18 surgically resected human GCA specimens were collected from the same hospitals as above. Of the 18 cases,13 were males with an average age of 60±8 years old and 5 females with an average age of 61±10 years old.
Biospy tissue:All the 18 endoscopic biopsy esophageal epithelium as normal control were collected from high-incidence regions. Of the 18 cases,9 were males with an average age of 48±10 years old and 9 females with an average age of 50±11 years old.
Paraffin-embedded tissue:All the 30 surgically resected human EC specimens were collected from the same hospitals as above. Of the 30 cases,18 were males with an average age of 57±8 years old and 12 females with an average age of 57±10 years old.
CC patients:All the 17 cases patients with CC in this study were from the same hospitals as above. Of the patients,12 were males and 5 were females, with a mean age of 59±7 years in male and 58±9 years in female. All the CC patients were conformed to primary SCC and GCA from the same patient by histopathology.
None of the patients had received chemotherapy and/or radiotherapy before operation. All the cases were confirmed as GCA or SCC respectively by histopathology. TNM staging was undertaken based on the criteria by AJCC (2002, AJCC).
2.2 Tissue collection and processing
Within 2 hours after surgical excision, the specimens were cut into two-halves longitudinally. Half of the specimen was fixed with 85% ethanol, paraffin-embedded, and sectioned at 5μm for histopathological evaluation and immunohistochemical analysis. The other part of the specimens was stored in the liquid nitrogen first and then transferred in a-80℃freezer for DNA extraction.
2.3 Methods
2.3.1 DNA extraction for frozen tissue
DNA extraction from the SCC and GCA pathology sample was performed with QIAamp DNA mini kit, QIAGEN company DNA mini kitTM (51306).
2.3.2 DNA extraction for paraffin-embedded tissue
Briefly,10-15 slides were deparaffinized in xylene and graded alcohol, then the lysis buffer (300 mmol/1 NaCl; 50 mmol/1 Tris.hCl pH 8.0; 0.2% SDS) was added into the tube with proteinase K (0.5 mg/ml), and the solution was incubated at 55℃overnight until it became clear. Then DNA was extracted using phenol/chloroform, precipitated with cold alcohol, and dissolved in ion-free water and stored at-20℃
2.3.3 Immunohistochemical analysis
Avidin-biotin-peroxidase complex (ABC) method was used to analyze the expression of p53, p16, p21WAF1 and MDM2. The classification of immunoreactivity was based on the immunostaining intensity (score as 0,1,2,3) and cell proportion of positive immunostaining (score as 0,1,2,3,4).
2.3.4 Identification for the purity and integrity of DNA samples
The DNA concentration was determined from its optical density. Quality of the extracted DNA was tested by PCR with beta-actin primer:5'-TCA CCC ACA CTG TGC CCA TC-3'and 5'-GAA CCG CTC ATT GCC AAT GG-3'.
2.3.5 HPV16 E6 amplification
The usable DNA went through PCR amplification using primer:5'-TCA AAA GCC ACT GTG TCC TG-3'and 5'-CGT GTT CTT GAT GAT CTG CA-3'targeting HPV-16 E6 gene under conditions at 95℃denaturing for 5 min; at 95℃denaturing for 1 min,55℃annealing for 1 min, and at 72℃prolonging for 1 min with 5 cycles; then at 95℃denaturing for 20s,55℃annealing for 30s, and at 72℃prolonging for 30s with 40 cycles; at 72℃prolonging for 5 min. The PCR product was about 120 bp. The plasmid containing full of length of HPV-16 genome as template was the positive control, and the water as template was the negative control.
2.3.6 Statistics
Chi-squared test and Kappa test were performed to compare the difference between groups.
3. RESULTS
HPV16 E6 amplification for 44 EC frozen tissues
All the forty-four extracted DNA samples showed good quality of DNA after PCR with beta-actin primer. After PCR amplification using HPV-16 E6 specific primer, HPV infection was found in tumor patients for 84.1% (37/44) in Linzhou. There was no association between HPV16 infection and gender, age, differentiation, lymph node metastasis and tumor staging.
HPV16 E6 amplification for 18 GCA frozen tissues
All the eighteen extracted frozen gastric cardia cancer samples showed good quality of DNA after PCR with beta-actin primer. PCR amplification using HPV-16 E6 specific primer, HPV infection was found in tumor patients for 44.4%(8/18) in Linzhou, risk incidence area of esophageal cancer. No association between HPV 16 infection and gender, age, differentiation, lymph node metastasis and tumor staging.
HPV16 E6 amplification for 18 biospy esophageal epithelium
Using the same method, HPV infection rate was found in endoscopic biopsy esophageal epithelium as normal control was collected from high-incidence regions for33.3%(6/18).
HPV16 E6 amplification for 30 paraffin-embedded
Using the same method, HPV infection rate was found in frozen sample of esophagus for 84.1% and 53.3%(16/30) from paraffin-embedded tissue, respectively. But comparing the positive rate of frozen and paraffin-embedded samples, the difference of HPV infection in DNA level was significant (P< 0.05).
HPV 16 E6 amplification for 17 CC
All the extracted DNA samples showed good quality of DNA after PCR with beta-actin primer. After PCR amplification using HPV-16 E6 specific primer, HPV infection was found in tumor patients from both tissue, with an infection rate of 47.1% (8/17) in SCC and 29.4% in GCA. There have no significant differences among them by statistics (P> 0.05).
Immunohistochemical staining for 17 CC
In CC patients, the positive immunohistochemical staining for p53, p16, p21WAF1 and MDM2 was observed both in SCC and GCA with different degrees. In SCC, the overexpression rates of p53 was the highest with 64.7%(11/17), followed by MDM2, p16 and p21WAF1 with 58.8%(10/17),41.2%(7/17) and 41.2%(7/17), respectively. In GCA, the overexpression rates of MDM2, p53, p16 and p21WAF1 were similar, with 70.6%(12/17),64.7%(11/17),58.8%(10/17) and 47.1%(8/17), respectively. Frequent immunostaining pattern for MDM2 and p53 was "Diffuse" and "Scatter". In p16, and p21WAF1, "Scatter" immunostaining pattern was predominant. High coincidence rates for MDM2, p53, p16 and p21WAF1 positive staining was observed in SCC and GCA from the same patients, and were 70.6%(12/17),64.7%(11/17),58.8%(10/17) and 47.1%(8/17), respectively.
Comparison for HPV infection and p53, p16, p21WAF1, MDM2 immunohistoche-mical staining in 17 CC
In CC patients, correction was analyed both in SCC and GCA with HPV infection and immunohistochemical staining for p53, p16, p21WAF1 and MDM2. In GCA, the correction of HPV infection and p53, p21WAF1, MDM2 were the highest for 1.0, followed by p16 for 0.338, respectively. In SCC, the correction of HPV infection and p53, p16 and p21WAF1, MDM2 were similar, with 0.335,0.335,1.0 and 0.637, respectively.
4. CONCLUSIONS
4.1 The prevalence of HPV16 in the esophageal cancer patients was higher than that gastric cardia cancer and healthy control subjects, which indicate that high-risk HPV plays an important role in the development of esophageal cancer of risk-incidence areas. Statistical analyses show that the prevalence of HPV is not dependent on gender, age, differentiation, lymph node metastasis and tumor staging.
4.2 The incidence of gastric cardia adenocarcinoma is also very high in this area, higher detection rate of HPV in gastric cardia adenocarcinoma indicate that HPV may be an aetiological factor in carcinogenesis of GCA.
4.3 HPV DNA was amplified with a high detection rate in endoscopic biopsy healthy subjects in risk incidence area show HPV as a tumor promoters preceding carcinogenesis progression.
4.4 30 cases of pair integrity esophageal cancer sample, the fresh(frozen) and paraffin- embedded tissues amplified using the same approach, the two organizations HPV16 E6 in the detection rate was 84.1% and 53.3%, respectively. The matching test, (P<0.01); the limitations of method itself should be excluded, paraffin-embedded tissues was significantly lower than fresh tissue. This may be fixed and all organizations in the process, the DNA breakdown, the formation of some of the smaller fragments.
4.5 The CC patients (the same individual), and HPV in the SCC in the detection rate of 47.1%(8/17), the detection rate was 29.4%(5/17) in the GCA. When comparing the two no significant difference (P> 0.05). HPV infection may suggest that the high incidence area of common participation in the esophagus and gastric cardia cancer process, the two have similar carcinogenic risk factors.
4.6 For CC patients, HPV infection and p53, p16, p21WAF1, MDM2 protein changes associated, Which p53, p21WAF1, MDM2 protein changes associated with more GCA, Suggest that the evolution of the process of carcinogenesis may have different molecular basis.
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