从贮存20年的微量细胞中提取DNA并做PCR分析的实验研究

目的:探讨从二十年前贮存的食管拉网微量细胞涂片中提取DNA并进行食管癌基因检测的方法和可行性,比较P53基因的突变在食管癌前期病变和正常食管上皮中的差异。方法:利用螯合型的离子交换树脂Chelex100作为介质,一步法从食管拉网脱落细胞提取DNA,应用PCR-SSCP方法和PCR-RFLP方法进行p53基因第5外显子及第7外显子的突变检测。结果:48例标本P53基因检测均获成功。食管上皮重度不典型增生细胞中发现有5例突变发生,均为p53基因第7外显子,而第5外显子未发现突变;正常食管鳞状上皮拉网脱落细胞中均未发现突变。检测到的5例突变中,其中有3例分别在10年、12年和14年后转变为食管癌。结论:Chelex100法能从微量细胞涂片中提取DNA,使对20年前食管拉网细胞涂片标本进行分子水平的检测成为可能。为食管癌前期病变的研究提供一种新的方法。p53基因的突变使食管上皮癌前期病变细胞具有了癌变趋势。     

从贮存20年的微量细胞中提取DNA并做PCR分析的实验研究

目的:探讨从二十年前贮存的食管拉网微量细胞涂片中提取DNA并进行食管癌基因检测的方法和可行性,比较P53基因的突变在食管癌前期病变和正常食管上皮中的差异.方法:利用螯合型的离子交换树脂Chelex100作为介质,一步法从食管拉网脱落细胞提取DNA,应用PCR-SSCP方法和PCR-RFLP方法进行p53基因第5外显子及第7外显子的突变检测.结果:48例标本P53基因检测均获成功.食管上皮重度不典型增生细胞中发现有5例突变发生,均为p53基因第7外显子,而第5外显子未发现突变;正常食管鳞状上皮拉网脱落细胞中均未发现突变.检测到的5例突变中,其中有3例分别在10年、12年和14年后转变为食管癌.结论:Chelex100法能从微量细胞涂片中提取DNA,使对20年前食管拉网细胞涂片标本进行分子水平的检测成为可能.为食管癌前期病变的研究提供一种新的方法.p53基因的突变使食管上皮癌前期病变细胞具有了癌变趋势.     

乳腺增生病p53基因第8外显子突变及p53蛋白表达

目的：探讨p53基因在乳腺癌发生早期的作用。方法：用免疫组化检测36例乳腺单纯增生，31例不典型增生，30例乳腺癌组织中p53蛋白表达，用PCR－RFLP检测p53基因第8外显子第278密码子突变。结果：乳腺单纯性增生，不典型增生和乳腺癌中p53蛋白表达率分别为0，22．6％，46．7％。p53基因第8外显子的突变率分别为0，3．2％，10．0％，均为杂合子突变。结论：乳腺癌不典型增生中存在p53蛋白表达和该基因第8外显子突变，该突变可能在乳腺增生病向乳腺癌进展过程中起一定作用。     

食管癌变过程中p53表达及其与细胞凋亡水平的关系

[目的]探讨食管癌变过程中食管鳞状上皮p53表达情况及其与凋亡水平的关系。[方法]824例食管黏膜活检组织（包括正常黏膜、异型增生和鳞癌）,行DNA末端标记（TUNEL法）检测及p53免疫组化检查。[结果]①轻—中度不典型增生组p53表达阳性率高于正常及炎症组和良性增生组（P=0.003）,低于重度不典型增生/鳞癌组（P=0.036）;良性增生组与轻—中度不典型增生组间无明显差异（P=0.192）;②正常及炎症组和良性增生组随年龄增长p53表达呈上升趋势（P〈0.01）;轻中—度不典型增生组及重度不典型增生组各年龄组间差异不明显;③随着p53表达的增加,上皮颗粒层细胞凋亡指数（AI）逐步下降（χ2=87.266,P=0.000）,基底层AI呈轻度增加趋势（χ2=34.253,P=0.000）。[结论]食管癌变过程中伴有p53基因的突变和颗粒层细胞凋亡水平的下降,且两者关系密切。     

p53及PCNA的异常表达在食管上皮增生和癌变过程中的意义

目的:研究食管上皮增生、不典型增生及原位癌中p53蛋白及增殖细胞核抗原(PCNA)的异常表达,探讨其在食管癌发生发展中的作用.方法:用免疫组化LSAB方法检测189例食管鳞癌癌旁上皮及原位癌中p53及PCNA的表达.结果:癌旁上皮中存在p53蛋白积聚,从上皮增生→不典型增生→原位癌,其阳性率依次为55 %、79 %和98 %(P＜0.01).PCNA的阳性强度也是依次递增.结论:p53蛋白的积聚在食管鳞癌癌前病变中既已存在,说明它是一个早期事件,在食管癌的发生中起一定的作用.p53及PCNA的异常表达可能成为判断食管上皮发生癌变或癌前病变的客观指标之一.     

p53基因在人食管诱发癌中的突变

目的：了解抑癌基因p53在人食管癌及其癌旁组织中的突变和致癌作用。方法：采用PCR-SSCP和DNA序列分析等方法对24例人食管鳞状细胞癌术后标本和21例移植到重度完全性免疫缺陷(severe：comlbined immunocleficient，SCID)小鼠并用或不用N-戊基-N-甲基亚硝基胺(N-amyrl-N-methylnitrosamine，AMN)处理的人食管正常组织标本，进行了p53基因外显子4～8的突变研究分析。结果：在食管癌组织及癌旁组织均检测出p53基因突变，其中食管癌组织标本中有18个突变，癌旁组织标本中有10个(10／17，58．8％)突变，主要的突变为G-A转换。在14例用AMN处理的人食管正常组织标本中出现6个(42．9％)突变；其中密码子283的突变为G-A转换。在人食管癌及其癌旁组织中p53基因的突变概率差异无统计学意义。结论：环境因素或内源性AMN可能是河南林县食管癌高发的主要原因之一。     

食管鳞状上皮细胞癌中P53基因突变的PCR-SSCP检测

应用PCR－SSCP银染技术检测了40例食管鳞状细胞癌p53基因第5、6、7和第8外显于的突变情况。结果显示：13例（32．5％）有p53基因突变，突变主要发生在第5和第8外显子，与癌分化程度无明显关系，但伴淋巴结癌转移病例中p53基因的突变率明显高于无转移者（P＜0．05）。本研究提示，p53基因突变存在于中、晚期食管癌中，并可能与食管鳞癌的进展与转移有关。     

维甲酸受体-β等肿瘤分子标志物在食管正常及异常组织中的表达及意义

目的 研究食管癌高发区人群的正常食管上皮、癌前病变及癌组织活检标本中,维甲酸受体-β(RAR-β)mRNA、p16、p53和Ki67蛋白的异常表达及其与食管癌发生的关系。方法 全组397例标本,其中食管正常黏膜组织25例,轻度不典型增生69例,中度不典型增生106例,重度不典型增生51例,原位癌78例,鳞状细胞癌68例。应用RNA原位杂交技术检测RAP-β mRNA的水平,应用免疫组织化学技术检测p16、p53和Ki67蛋白的表达情况。结果 在正常黏膜、轻度、中度、重度不典型增生、原位癌及鳞状细胞癌组织中,RAR-β mRNA表达检出率分别为96．0%、89．9%、67．9％、68．6％、62．8％和63．2％;p16表达检出率分别为88．0％、71．0％、64．2％、51．0%、53．8％和52．9%;p53异常表达检出率分别为4．0％、39．1％、57．5%、52．9％、67．9％和69．1％;Ki67异常表达检出率为0、40．6％、61．3％、58．8%、59．0％和75．0%。结论 4种肿瘤生物学标志物在食管上皮中、重度不典型增生和原位癌组织中的表达,未出现明显差异,而只表现为形态学上的差异。     

细胞图像分析预测食管上皮增生转归的研究

目的　探讨食管癌前期病变细胞核的染色质特征与癌变的关系,并对其癌变进行早期预测。方法　标本取自1983 年林县河顺乡食管细胞学普查。鳞状上皮重度增生93 例,轻度增生122例。所有标本均为食管拉网涂片,巴氏染色。图像输入设备是Axiomat 图像分析系统,由VAX4000500计算机控制,idl 软件。每一样本随机选择100 以上保存完好的鳞状上皮中层细胞,用100 ×物镜进行细胞核的测量。结果　从100 多个染色质形态、光密度和纹理的特征中,选出15 个有意义的特征进行判断。93 例重度增生3 年、5 年和9 年后癌变的病例作为高分辨率细胞分析样本,细胞图像分析判断为癌变的分别为18 例、12 例和6 例,判断正确率分别为75 .0 % (18/24) 、85 .7 % (12/14) 和85 .7 % (6/7) ;122 例轻度增生3 年、5 年和9 年后癌变的病例,细胞图像分析判断为癌变的分别为15 例、10 例和10 例,判断正确率分别为93 .8 % (15/16) 、76 .9 % (10/13) 和83 .3 % (10/12) 。结论　利用高分辨率细胞图像分析系统提取细胞核的特征能够区别癌变组和非癌变组,并可对癌前病变进行癌变的早期预测,在肿瘤预防研究中可以作为代用终期生物学指标之一。     

p53基因在人食管诱发癌中的突变

目的 :了解抑癌基因p5 3在人食管癌及其癌旁组织中的突变和致癌作用。方法 :采用PCR SSCP和DNA序列分析等方法对 2 4例人食管鳞状细胞癌术后标本和 2 1例移植到重度完全性免疫缺陷 (severecombinedimmunodefi cient,SCID)小鼠并用或不用N -戊基 -N -甲基亚硝基胺 (N amyl N methylnitrosamine ,AMN)处理的人食管正常组织标本 ,进行了p5 3基因外显子 4～ 8的突变研究分析。结果 :在食管癌组织及癌旁组织均检测出p5 3基因突变 ,其中食管癌组织标本中有 18个突变 ,癌旁组织标本中有 10个 ( 10 /17,5 8 8% )突变 ,主要的突变为G A转换。在 14例用AMN处理的人食管正常组织标本中出现 6个 ( 4 2 9% )突变 ;其中密码子 2 83的突变为G A转换。在人食管癌及其癌旁组织中p5 3基因的突变概率差异无统计学意义。结论 :环境因素或内源性AMN可能是河南林县食管癌高发的主要原因之一     

P53 expression in esophageal dysplasia - a possible biomarker for carcinogenesis of esophageal squamous-cell carcinoma

The tumor suppressor gene product p53 has been detected in a high percentage of esophageal squamous cell carcinoma. To evaluate the role of this protein in carcinogenesis, we examined the p53 overexpression both in esophageal dysplasia and in esophageal squamous cell carcinoma in the same patients. Using anti-p53 antibodies pAb1801 and CM-1, we analyzed immunohistochemically 36 dysplastic lesions from 36 patients with esophageal cancer. Nuclear p53 was detected in 14 of 36 dysplasias (39%). From mild to moderate to severe dysplasia, p53 positivity showed tendency to increase in number. Seventeen of the 36 squamous cell carcinomas showed p53 expression (47%). There was a significant concurrent p53 expression in esophageal dysplasia and its related squamous cell carcinoma (p=0.00345). These results indicate that p53 mutation is closely associated with the initiation of this cancer.     

Frequent mutation of p16(CDKN2A) exon 1 during rat tongue carcinogenesis induced by 4-nitroquinoline-1-oxide

In this study we explored the mutation types of p16(CDKN2A) exon 1 and the corresponding frequencies in experimental rat tongue carcinogenesis. Twenty barrier Sprague-Dawley (SD) rats were divided into the control (n = 5) and experimental group (n = 15), to which 4-nitroquinoline-1-oxide (4-NQO) in drinking water was administered. Two samples of normal, three samples of moderate/severe dysplasia and four samples of invasive squamous cell carcinoma lesions were selected following strict histopathological examination in double-blind manner. The PCR products of p16(CDKN2A) exon 1 amplified from these tissues were sequenced. Point mutations of p16(CDKN2A) exon 1 were found in all of the precancerous and cancerous lesions. Half of the mutations were detected on guanine (G). Twenty mutations, including a missense mutation of the start codon resulting in alternative reading frame of p16(CDKN2A) exon 1, were also identified. These preliminary results suggested that mutation of p16(CDKN2A) exon 1 might be an early molecular event of rat tongue carcinogenesis induced by 4NQO and G was the mutation hotspot.     

p53 Gene mutation and genetic instability in superficial multifocal esophageal squamous cell carcinoma

Tumor multicentricity is occasionally observed in esophageal squamous cell carcinoma (SCC). We studied five surgically resected superficial multifocal esophageal SCCs for p53 gene mutation and genetic instability, using DNA extracted from microdissected areas. A total of 38 target areas (TAs) were analyzed in SCC, dysplasia, basal cell hyperplasia (BCH) and normal squamous epithelium. Analysis of the replication error (RER) at 10 microsatellite loci showed microsatellite instability in all TAs, as well as in normal squamous epithelium. p53 gene mutation was identified in 28.9% (11/38 TAs). All cases showed a common missense mutation in exon 8 at codon 273 (CGT-->CAT, Arg-->His), which was DNA contact mutation in the S10 beta strand. In association with microsatellite alterations, 7 of 9 TAs with p53 mutation in exon 8 at codon 273 also showed loss of heterozygosity (LOH) of p53 gene. LOH of p53 gene was detected in 83.8% (31/37 TAs). LOH at D2S123 on 2p16 near MSH2 gene and at D3S1611 on 3p22 near MLH1 gene was detected in 65.4% (17/26) and 71.4% (10/14) TAs, respectively. Frequencies of LOH at p53 and D2S123 were similar in non-cancerous areas and SCCs. LOH of p53 and D2S123 were found in 50% (5/10 TAs) of non-cancerous areas and 60% (9/15 TAs) of SCCs. Our results suggest that genetic instability induces esophageal tumor multicentricity, and that p53 gene contact mutation together with LOH are early events of the multistage carcinogenesis of multifocal primary esophageal SCC.     

Study of p53 gene alteration as a biomarker to evaluate the malignant risk of Lugol-unstained lesion with non-dysplasia in the oesophagus

Mutations of the p53 gene are detected frequently in oesophageal dysplasia and cancer. It is unclear whether Lugol-unstained lesions (LULs) with non-dysplastic epithelium (NDE) are precursors of oesophageal squamous cell carcinoma (ESCC). To study the genetic alterations of NDE in the multistep process of oesophageal carcinogenesis, we determined the relationship between p53 mutations and LULs-NDE. Videoendoscopy with Lugol staining was performed prospectively in 542 oesophageal cancer-free subjects. Lugol-unstained lesions were detected in 103 subjects (19%). A total of 255 samples, including 152 LULs (NDE, 137; dysplasia, 15) and 103 paired samples of normal staining epithelium, were obtained from 103 subjects. After extraction of DNA and polymerase chain reaction analysis, direct sequencing method was applied to detect mutations of the p53 gene. The p53 mutation was detected in five of 137 samples with LULs-NDE (4%) and in five of 15 samples with dysplasia (33%). A hotspot mutation was found in 20% of LULs-NDE with p53 mutation and in 40% of dysplasia with p53 mutation. In contrast, no p53 mutations were found in 103 paired NDE samples with normal Lugol staining. In biopsy samples from oesophageal cancer-free individuals, the p53 missense mutations containing a hotspot mutation were found in NDE, which was identified as an LUL. These findings suggest that some LULs-NDE may represent the earliest state of oesophageal squamous cell carcinoma in Japanese individuals.     

Role of p53 gene mutations in human esophageal carcinogenesis: results from immunohistochemical and mutation analyses of carcinomas and nearby non-cancerous lesions

In order to characterize p53 alterations in esophageal cancer and to study their roles in carcinogenesis, we performed gene mutation and immunohistochemical analysis on 43 surgically resected human esophageal specimens, which contain squamous cell carcinoma (SCC) and adjacent non-cancerous lesions, from a high-incidence area of Linzhou in Henan, China. A newly developed immunohisto-selective sequencing (IHSS) method was used to enrich the p53 immunostain-positive cells for mutation analysis. p53 gene mutations were detected in 30 out of 43 (70%) SCC cases. Among 29 SCC cases that were stained positive for p53 protein, 25 (86%) were found to contain p53 mutations. In five cases of SCC with homogeneous p53 staining, the same mutation was observed in samples taken from four different positions of each tumor. In a well differentiated cancer nest, p53 mutation was detected in only the peripheral p53-positive cells. In tumor areas with heterogeneous p53 staining, either the area stained positive for p53 had an additional mutation to the negatively stained area or both areas lacked any detectable p53 mutation. In the p53-positive non-cancerous lesions adjacent to cancer, p53 mutations were detected in seven out of 16 (47%) samples with basal cell hyperplasia (BCH), eight out of 12 (67%) samples with dysplasia (DYS), and six out of seven (86%) samples with carcinoma in situ (CIS). All mutations found in lesions with DYS and CIS were the same as those in the nearby SCC. In seven cases of BCH containing mutations, only three had the same mutations as the nearby SCC. The results suggest that p53 mutation is an early event in esophageal carcinogenesis occurring in most of the DYS and CIS lesions, and cells with such mutations will progress to carcinoma, whereas the role of p53 mutations in BCH is less clear.     

p53 overexpression in normal and dysplastic tissues adjacent to p53 negative squamous cell carcinomas of the head and neck

p53 overexpression was present in the normal or dysplastic epithelium, but absent in the adjacent invasive cancers of five patients with head and neck squamous cell carcinomas (HNSCC), when p53 immunostaining (IHC) was performed. In three of the five p53 immunoreactive dysplasias and adjacent p53 negative invasive cancers single stranded conformation polymorphism (SSCP) results from exon 7 and 8 were also obtained. Bandshifts in exon 7 were detected in two dysplasias, and bandshifts in exon 8 were found in a third. Sequencing of exon 7 in the first dysplasia with bandshift indicated a deletion of codon 241-242 (loss of CT) resulting in a frame shift. In the second dysplasia with bandshift a mutation was observed in codon 244 resulting in a Gly-->Arg substitution in the protein sequence. In the adjacent IHC p53 negative invasive cancer lesions, no bandshifts could be observed by SSCP, and sequencing did not reveal any mutated p53. WAF1/p21 (IHC) expression was assayed to study p53 function. Image cytometry (ICM) DNA analysis, estimating genetic instability, showed progress in DNA aberration for invasive cancer lesions as compared with the dysplasias. Human papillomavirus (HPV DNA) was not detected by a polymerase chain reaction (PCR) in any of the five cancers thus excluding possible p53 degradation caused by HPV protein. In conclusion, the finding of p53 mutations in mild, moderate, and severe dysplasia indicates that p53 mutation, not only p53 immunoreactivity, can be an early event in HNSCC carcinogenesis. The lack of p53 immunoreactivity in the invasive cancers adjacent to p53 positive dysplasias could possibly be attributed to loss of the mutant allele, or clonal heterogeneity.     

Alterations of p53, cyclin D1 and pRB expression in the carcinogenesis of esophageal squamous cell carcinoma

Many molecular alterations occur in esophageal carcinogenesis; however, little is known about the molecular genetic events responsible for the development of carcinoma. We investigated the expression of ki67, p53, cyclin D1 and pRB in 105 biopsy specimens using immunohistochemistry from iodine unstained lesions as indicators of carcinogenesis of the esophagus. Also, the genetic alternation of esophageal dysplasia from patients with accompanying esophageal squamous cell carcinoma (ESCC) was examined to study the evidence for field carcinogenesis in the esophagus. The expression of p53, cyclin D1 and pRB was detected in 31, 0 and 51.7% respectively of mild dysplasia; 40, 0 and 70% of moderate dysplasia; 40, 20 and 70% of severe dysplasia; and 48, 32 and 80% of carcinoma specimens. p53 expression was significantly increased in mild dysplasia, whereas cyclin D1 and pRB expression were significantly increased in carcinoma as compared to both normal epithelium and esophagitis. The ki67 LI and the rate of p53 expression were significantly higher in dysplasia with ESCC than in dysplasia without ESCC. Ki67, p53, cyclin D1 and pRB expression may be useful biomarkers for assessing the risk of developing esophageal cancer. Dysplasia observed at screening for secondary lesions has a highly malignant potential and careful follow-up studies are required.     

Hypermethylation-associated inactivation of retinoic acid receptor beta in human esophageal squamous cell carcinoma.

PURPOSE: The purpose of this study was to investigate the mechanism of altered retinoic acid receptor beta (RARbeta) expression during esophageal squamous carcinogenesis. EXPERIMENTAL DESIGN: Samples were collected from Linzhou, China. The hypermethylation of CpG islands in the promoter region of the RARbeta gene was examined by methylation-specific PCR in human esophageal squamous cell carcinoma (ESCC) samples, as well as in neighboring tissues with normal epithelium, basal cell hyperplasia, and dysplasia. RARbeta mRNA expression was determined by in situ hybridization. The DNA methyltransferase inhibitor 2'-deoxy-5-azacytidine was used to treat the ESCC cell line, and the DNA hypermethylation status and mRNA expression level were examined. RESULTS: Two of 17 (12%) normal, 9 of 21 basal cell hyperplasia (43%), 7 of 12 dysplasia (58%), and 14 of 20 ESCC (70%) samples had hypermethylation of the RARbeta promoter region. The loss of RARbeta mRNA expression was highly concordant with RARbeta promoter CpG island hypermethylation when individual samples were considered in the correlation analysis. Good statistical correlation between hypermethylation and loss of RARbeta expression was revealed. Frequencies of hypermethylation appeared to increase with the progression of carcinogenesis. In samples from the same patients, if hypermethylation was detected in earlier lesions, it was usually observed in more severe lesions. In the ESCC cell line KYSE 510, 2'-deoxy-5-azacytidine partially reversed CpG island hypermethylation and restored RARbeta mRNA expression. CONCLUSIONS: The results suggest that hypermethylation of RARbeta promoter region is an important mechanism for RARbeta gene silencing in esophageal squamous carcinogenesis.