食管和贲门癌变机制：河南高低发区人群普查、随访和p53-Rb基因变化
摘要
研究背景，目的和意义
河南省林州(原林县)及其毗邻的安阳，辉县等地是世界上食管癌发病率和死亡率最高的地区。食管癌仍然是该地区肿瘤相关死亡的主要原因。食管癌的主要特征之一是其与贲门癌有相似的流行病学特征。值得提出的是，该地区同一个体同时或先后发生食管鳞状上皮癌(SCC)和贲门腺癌(GCA)的现象，又称双源癌(CC)，并非罕见，发病率约为0.4-2.5％。GCA的研究资料甚少，发病机制尚不清楚。SCC和GCA的预后极差，中晚期患者生存率仅10％左右。该地区此二种肿瘤的发生率和死亡率目前均未得到有效控制，其原因主要是缺乏早期诊断的敏感指标和方法，缺乏有效的防治措施。过去研究资料提示，食管和贲门上皮癌变是一个多阶度演进过程。SCC和GCA的易感人群的早期特征是上皮细胞增生异常，食管上皮增生异常的形态学表现主要是基底细胞过度增生(BCH)、间变(DYS)和原位癌(CIS)，贲门上皮主要表现为肠上皮化生和间变，这些病变被认为是食管和贲门的癌前病变。癌前病变的显著特征之一是具有双向发展不稳定特性，即，这些病变可维持在某一阶段多年不变，或发展成更重的病变，甚至可退回到正常或较轻的病变。很显然，单纯从形态学角度，难以解释这种现象，本研究的思路正是在老一代科学家工作的基础上，通过对河南SCC和GCA高发区无症状居民进行内镜活检和拉网细胞学普查，寻找早期癌和各级癌前病变患者，并对其进行随访和Ⅱ级预防，进而建立同一个体，不同时间，不同阶段大样本资料库，在此基础上，进一步利用分子生物学技术研究这些不同病变组织的
浙江大学博士学位论文
分子变化[p53一Rb系统变化，(包括p53，Rb，pls，pl6，pl4，waflpZI，eyelinnl，
PCNA等)，与环境致病危险因素相关的酶基因多态改变(包括CYPIAI，IEI，
MEH，GsTMITI，p]系)和全基因组杂交(CGH)]，最终目的是阐明食管和贪
门癌变机制，建立用于高危人群检测和早期诊断的生物指标和手段，为食管责门
癌防治提供重要的理论基础和手段，最终降低食管和贵门癌的发病率和死亡率。
材料与方法
研究对象
本研究所有研究对象均来自河南食管和贵门癌高发区(n=11，230)。无症状
患者主要来自食管和责门早期癌人群普查，所有研究对象均未作任何人为筛选。
35岁以上的当地居民(研究现场)均被邀请参加早期癌普查和干预阻断。这些
人群的详细资料，包括年龄，性别等会在后面章节中详加描述。所有研究对象均
征得本人同意。本研究中，SCC和GCA患者也来自同一地区。所有收集的手术
切除标本术前患者均未作化疗或放疗。患者详情见随后相关章节。
内镜和猫膜活检检查
每例复检者，均在食管中，下段(距门齿30一32cm和食管/责门交界线上2一3cm
处)和贪门部各取一块活检组织，并在肉眼可见的病灶处随机取材。所有活检组
织均迅速经85%酒精固定，石蜡包埋和5林m连续切片，切片用于HE染色，病
理诊断，免疫组化和ONA提取等。
拉网细胞学检查
每一受检者均嘱随吞咽动作咽下食管拉网细胞采集器，至责门下方，充气后，
一边逐渐放气，一边轻轻拉出，将网上拉出的上皮细胞涂2一3张涂片，并迅速酒
精固定，巴氏染色。
组织和细胞学诊断标准
食管和贵门组织和细胞学诊断标准采用我们实验室以往建立的标准，根据组
浙江大学博士学位论文
织结构和细胞特征，将食管上皮分为正常，BCH，
拉网细胞分为正常，轻增，重增I级，重增H级
常，CGS，C^q DYs和GCA.
DYS、CIS和
近癌和癌，
SCC，将食管上皮
将责门上皮分为正
随访设计
采用重复活检和细胞学检查进行随访。随访进行中，不定期对随访者进行访
问，并作详细记录，包括有无特殊不适，其他疾病，生活习惯改变等。
CGH
CGH的主要工作原理:SpectlumGreen一dU仰标记食管癌和责门癌DNA，
SpectrumRed一dUTP标记正常基因组DNA，二者以1:1比例混合后制备成探针，
同时与正常人外周血的有丝分裂中期染色体进行原位杂交。在荧光显微镜下肿瘤
DNA呈绿色，正常组织DNA呈红色;通过检测两种颜色的荧光强度，根据两种
颜色的比率情况来显染色体的结构状况，如发现两种颜色比率改变，则说明该区
域存在DNA序列的缺失或扩增。CGH的主要步骤是:应用切口平移法分别用
speetrum一Green一dUTp和speetrum Red一dUTp(Vysis，Downers Grove)标记1陀
肿瘤组织DNA和l林g正常参照组DNA。用标记好的肿瘤基因组DNA探针和正
常参照组探针ZOOng和10林g人Cot一IDNA溶于10ul杂交缓冲液中(体积分数为
50%甲胺，Zxssc，pH7.0) 75oc变性5分钟。制备好的中期分裂像玻片，
100林g/mlRNase处理37oCI小时，70%甲酸胺变性液75“C，变性5分钟，ZxSSC
各5分钟。杂交混合液滴于片上，湿盒内37oC孵育3天。杂交后玻片用
O.4xSSC/0.3%NP40洗液75“C洗片2分钟，然后用ZxSSC/0.1%NP40室温下洗片
2分钟，洗片后用l卜、g/川的DAPI40闪进行染色。
IHC
利用卵蛋白一生物素一辣根过氧化氢酶复合物法(ABc)，检测组织中p53，
PCNA，Rb， WaflP21，Bcl一2，C一myc，TGFB等抗原。详细步骤见随后相应章节
描试_氛幻为沿对昭幻
Linzhou (formerly Linxian) and nearby counties in Henan province, northern China have been well recognized as the highest incidence area for esophageal squamous cell carcinoma (SCC) in the world. SCC remains the leading cause of cancer-related deaths in these areas. A remarkable epidemiological characteristic for SCC is the occurring together with gastric cardia adenocarcinoma (GCA) in the same high-incidence area. It is noteworthy that the concurrent cancers from esophagus and gastric cardia in same patient (CC) is not uncommon in this area, with an incidence of 0.4-2.5%. GCA is still an understudied subject and largely unknown for the mechanism of carcinogenesis. Moreover, the prognosis for both SCC and GCA is very poor; the five years survival rate for these two cancers at the late and advanced stages is only 10%. Lacking of early diagnostic biomarker as well as the deficiency of effective and specific treatment and prevention leads to the poor prognosis and higher
mortality for SCC and GCA in these areas. Accumulated data have indicated that carcinogenesis both for SCC and GCA is a multistage progress. The early indicator for the subjects predisposed to SCC and GCA is the abnormal proliferation of epithelial cells, morphologically manifested as basal cell hyperplasia (BCH), dysplasia (DYS) and carcinoma in situ (CIS), which could be considered as precancerous lesions for SCC. Intestinal metaplasia and DYS have been recognized as precancerous lesions for gastric cardia carcinogenesis. Precancerous lesions have potential of reversibility, i.e., they may stay in the same satge for couple of years, or progress to more severe lesions, even return to normal. Apparently, it is limited to explain this phenomenon in morphological level. To further elucidate the possible mechanisms of SCC and GCA carcinogenesis, the present study was designed to establish data bank through endoscopic biopsy and balloon cytology screening, follow-up and intervention on subjects at high-incidence area in Henan and to determine the alterations of protein files (p53-Rb system, including p53, Rb, p16, p14, p15, wafl, Cyclin Dl, PCNA, etc) and genetic changes, polymorphisms of CYP1A1, CYP2E1, mEH, GSTM1, GSTT1 and GSTP1, comparative genomic hybridization (CGH).
MATERIALS AND METHODS
Subjects:
All the subjects enrolled in this study were from Linzhou and nearby counties in Henan, the highest incidence area for both SCC and GCA (n=l 1,230). The symptom-free subjects were those from mass survey for early detection of SCC and GCA, there was no selection process involved. All the inhabitants in these areas with
age of over 35 years were invited for screening and intervention trail. The detailed information for these subjects including age and sex was described in each sections followed. The patients with SCC and GCA in this study were also from the same areas. Surgically resected specimens were collected from those without chemotherapy or radiotherapy before operation. The detailed information for the patients was addressed in each section.
Endoscopy and Biopsies Examinations
Two biopsy samples were taken at standard positions, one each from the middle third (30-32cm from incisor teeth) and the lower third (2-3cm above the junction of the esophagus and cardia) of the esophagus. Another one was taken at gastric cardia. Additional biopsies were taken when there were macroscopic lesions. The biopsy specimens were fixed in 85% alcohol, embedded in paraffin, and sectioned at 5 m. Balloon cytological examination
Each subject was asked to swallow the balloon, and the balloon attached to the tube was gently passed down the esophagus to the level of the gastric cardia (50 cm from the front teeth). The distended balloon was gently withdrawn, with some release of air if resistance was encountered. Three smears were made from each person by rolling the balloon on glass slides and immediately fixing in 95% alcohol. All smears were stained with the Papanicolaou method and examined under light microscope. Criteria for histology and cytology
河南省林州(原林县)及其毗邻的安阳，辉县等地是世界上食管癌发病率和死亡率最高的地区。食管癌仍然是该地区肿瘤相关死亡的主要原因。食管癌的主要特征之一是其与贲门癌有相似的流行病学特征。值得提出的是，该地区同一个体同时或先后发生食管鳞状上皮癌(SCC)和贲门腺癌(GCA)的现象，又称双源癌(CC)，并非罕见，发病率约为0.4-2.5％。GCA的研究资料甚少，发病机制尚不清楚。SCC和GCA的预后极差，中晚期患者生存率仅10％左右。该地区此二种肿瘤的发生率和死亡率目前均未得到有效控制，其原因主要是缺乏早期诊断的敏感指标和方法，缺乏有效的防治措施。过去研究资料提示，食管和贲门上皮癌变是一个多阶度演进过程。SCC和GCA的易感人群的早期特征是上皮细胞增生异常，食管上皮增生异常的形态学表现主要是基底细胞过度增生(BCH)、间变(DYS)和原位癌(CIS)，贲门上皮主要表现为肠上皮化生和间变，这些病变被认为是食管和贲门的癌前病变。癌前病变的显著特征之一是具有双向发展不稳定特性，即，这些病变可维持在某一阶段多年不变，或发展成更重的病变，甚至可退回到正常或较轻的病变。很显然，单纯从形态学角度，难以解释这种现象，本研究的思路正是在老一代科学家工作的基础上，通过对河南SCC和GCA高发区无症状居民进行内镜活检和拉网细胞学普查，寻找早期癌和各级癌前病变患者，并对其进行随访和Ⅱ级预防，进而建立同一个体，不同时间，不同阶段大样本资料库，在此基础上，进一步利用分子生物学技术研究这些不同病变组织的
浙江大学博士学位论文
分子变化[p53一Rb系统变化，(包括p53，Rb，pls，pl6，pl4，waflpZI，eyelinnl，
PCNA等)，与环境致病危险因素相关的酶基因多态改变(包括CYPIAI，IEI，
MEH，GsTMITI，p]系)和全基因组杂交(CGH)]，最终目的是阐明食管和贪
门癌变机制，建立用于高危人群检测和早期诊断的生物指标和手段，为食管责门
癌防治提供重要的理论基础和手段，最终降低食管和贵门癌的发病率和死亡率。
材料与方法
研究对象
本研究所有研究对象均来自河南食管和贵门癌高发区(n=11，230)。无症状
患者主要来自食管和责门早期癌人群普查，所有研究对象均未作任何人为筛选。
35岁以上的当地居民(研究现场)均被邀请参加早期癌普查和干预阻断。这些
人群的详细资料，包括年龄，性别等会在后面章节中详加描述。所有研究对象均
征得本人同意。本研究中，SCC和GCA患者也来自同一地区。所有收集的手术
切除标本术前患者均未作化疗或放疗。患者详情见随后相关章节。
内镜和猫膜活检检查
每例复检者，均在食管中，下段(距门齿30一32cm和食管/责门交界线上2一3cm
处)和贪门部各取一块活检组织，并在肉眼可见的病灶处随机取材。所有活检组
织均迅速经85%酒精固定，石蜡包埋和5林m连续切片，切片用于HE染色，病
理诊断，免疫组化和ONA提取等。
拉网细胞学检查
每一受检者均嘱随吞咽动作咽下食管拉网细胞采集器，至责门下方，充气后，
一边逐渐放气，一边轻轻拉出，将网上拉出的上皮细胞涂2一3张涂片，并迅速酒
精固定，巴氏染色。
组织和细胞学诊断标准
食管和贵门组织和细胞学诊断标准采用我们实验室以往建立的标准，根据组
浙江大学博士学位论文
织结构和细胞特征，将食管上皮分为正常，BCH，
拉网细胞分为正常，轻增，重增I级，重增H级
常，CGS，C^q DYs和GCA.
DYS、CIS和
近癌和癌，
SCC，将食管上皮
将责门上皮分为正
随访设计
采用重复活检和细胞学检查进行随访。随访进行中，不定期对随访者进行访
问，并作详细记录，包括有无特殊不适，其他疾病，生活习惯改变等。
CGH
CGH的主要工作原理:SpectlumGreen一dU仰标记食管癌和责门癌DNA，
SpectrumRed一dUTP标记正常基因组DNA，二者以1:1比例混合后制备成探针，
同时与正常人外周血的有丝分裂中期染色体进行原位杂交。在荧光显微镜下肿瘤
DNA呈绿色，正常组织DNA呈红色;通过检测两种颜色的荧光强度，根据两种
颜色的比率情况来显染色体的结构状况，如发现两种颜色比率改变，则说明该区
域存在DNA序列的缺失或扩增。CGH的主要步骤是:应用切口平移法分别用
speetrum一Green一dUTp和speetrum Red一dUTp(Vysis，Downers Grove)标记1陀
肿瘤组织DNA和l林g正常参照组DNA。用标记好的肿瘤基因组DNA探针和正
常参照组探针ZOOng和10林g人Cot一IDNA溶于10ul杂交缓冲液中(体积分数为
50%甲胺，Zxssc，pH7.0) 75oc变性5分钟。制备好的中期分裂像玻片，
100林g/mlRNase处理37oCI小时，70%甲酸胺变性液75“C，变性5分钟，ZxSSC
各5分钟。杂交混合液滴于片上，湿盒内37oC孵育3天。杂交后玻片用
O.4xSSC/0.3%NP40洗液75“C洗片2分钟，然后用ZxSSC/0.1%NP40室温下洗片
2分钟，洗片后用l卜、g/川的DAPI40闪进行染色。
IHC
利用卵蛋白一生物素一辣根过氧化氢酶复合物法(ABc)，检测组织中p53，
PCNA，Rb， WaflP21，Bcl一2，C一myc，TGFB等抗原。详细步骤见随后相应章节
描试_氛幻为沿对昭幻
Linzhou (formerly Linxian) and nearby counties in Henan province, northern China have been well recognized as the highest incidence area for esophageal squamous cell carcinoma (SCC) in the world. SCC remains the leading cause of cancer-related deaths in these areas. A remarkable epidemiological characteristic for SCC is the occurring together with gastric cardia adenocarcinoma (GCA) in the same high-incidence area. It is noteworthy that the concurrent cancers from esophagus and gastric cardia in same patient (CC) is not uncommon in this area, with an incidence of 0.4-2.5%. GCA is still an understudied subject and largely unknown for the mechanism of carcinogenesis. Moreover, the prognosis for both SCC and GCA is very poor; the five years survival rate for these two cancers at the late and advanced stages is only 10%. Lacking of early diagnostic biomarker as well as the deficiency of effective and specific treatment and prevention leads to the poor prognosis and higher
mortality for SCC and GCA in these areas. Accumulated data have indicated that carcinogenesis both for SCC and GCA is a multistage progress. The early indicator for the subjects predisposed to SCC and GCA is the abnormal proliferation of epithelial cells, morphologically manifested as basal cell hyperplasia (BCH), dysplasia (DYS) and carcinoma in situ (CIS), which could be considered as precancerous lesions for SCC. Intestinal metaplasia and DYS have been recognized as precancerous lesions for gastric cardia carcinogenesis. Precancerous lesions have potential of reversibility, i.e., they may stay in the same satge for couple of years, or progress to more severe lesions, even return to normal. Apparently, it is limited to explain this phenomenon in morphological level. To further elucidate the possible mechanisms of SCC and GCA carcinogenesis, the present study was designed to establish data bank through endoscopic biopsy and balloon cytology screening, follow-up and intervention on subjects at high-incidence area in Henan and to determine the alterations of protein files (p53-Rb system, including p53, Rb, p16, p14, p15, wafl, Cyclin Dl, PCNA, etc) and genetic changes, polymorphisms of CYP1A1, CYP2E1, mEH, GSTM1, GSTT1 and GSTP1, comparative genomic hybridization (CGH).
MATERIALS AND METHODS
Subjects:
All the subjects enrolled in this study were from Linzhou and nearby counties in Henan, the highest incidence area for both SCC and GCA (n=l 1,230). The symptom-free subjects were those from mass survey for early detection of SCC and GCA, there was no selection process involved. All the inhabitants in these areas with
age of over 35 years were invited for screening and intervention trail. The detailed information for these subjects including age and sex was described in each sections followed. The patients with SCC and GCA in this study were also from the same areas. Surgically resected specimens were collected from those without chemotherapy or radiotherapy before operation. The detailed information for the patients was addressed in each section.
Endoscopy and Biopsies Examinations
Two biopsy samples were taken at standard positions, one each from the middle third (30-32cm from incisor teeth) and the lower third (2-3cm above the junction of the esophagus and cardia) of the esophagus. Another one was taken at gastric cardia. Additional biopsies were taken when there were macroscopic lesions. The biopsy specimens were fixed in 85% alcohol, embedded in paraffin, and sectioned at 5 m. Balloon cytological examination
Each subject was asked to swallow the balloon, and the balloon attached to the tube was gently passed down the esophagus to the level of the gastric cardia (50 cm from the front teeth). The distended balloon was gently withdrawn, with some release of air if resistance was encountered. Three smears were made from each person by rolling the balloon on glass slides and immediately fixing in 95% alcohol. All smears were stained with the Papanicolaou method and examined under light microscope. Criteria for histology and cytology
引文
1. Dawsey SM, Quiong S, Nieberg RK, Liu S-F, English SA, Cao J, Zhou B, Wang GQ, Lewin KJ, Liu F-S, Roth M J, Taylor PR: Studies of esophageal balloon cytology in Linxian, China. Cancer Epidemiol Biomark Prevent 1997; 6: 121-130
2. Li JY: Epidemiology of esophageal cancer in China. Natl Cancer Inst Monogr 1982; 62: 113-130
3. Koss LG: Cytologic diagnosis of oral, esophageal, and peripheral lung cancer. J Cell Biochem (suppl 17F) 1993; 66-81
4. Dawsey SM, Yu, Yaylor PR, Li JY, Shen Q, Shu Y J, Liu SF, Zhao HZ, Cao SG, Wangt GQ, Liu FS, Blot WJ, Li B: Esophageal cytology and subsequent risk of esophageal cancer: A prospective follow-up study from Linxian, China. Acta Cytol 1994; 38:183-192
5. Shen Q, Liu SF, Dawsey SM, Cao J, Wang DY, Cao SG, Zhao HZ, Li GY, Taylor PR, Guo WD, Liu FS, Blot WJ, Li B: Cytologic screening for esophageal cancer: Results from 12,877 subjects from a high-risk population in China. Int J Cancer 1993; 54:185-188
6. Lazarus C, Jaskiewicz K, Sumeruk RA, Nainkin J: Brush cytology technique in the detection of esophageal carcinoma in the asymptomatic, High-risk subject: A pilot suvey. Cytopathology 1992; 3:291-296
7. Liu SF, Shen Q, Dawsey SM, Wang GQ, Nieberg RK, Wang ZY, Weiner M, Zhou B, Cao J, Yu Y, Guo WD, Li JY, Blot W J, Li B, Taylor PR: Esophageal balloon cytology and subsequent risk of esophageal and gastric-cardia cancer in a high-risk Chinese population, lnt J Cancer 1994; 57:775-780
8. Sepehr A, Razavi P, Saidi F, Salehian P, Rahmani M, Shamshiri A: Esophageal exfoliative cytology samplers: A comparison of three types. Acta cytol 2000; 44:797-804
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