胃癌前病变APC抑癌基因原位杂交光镜及电镜观察

目的 探讨胃癌前病变ＡＰＣ基因异常表达及其意义。方法 应用光镜及电镜原位杂交对胃癌前病变ＡＰＣ基因进行细胞和超微水平观察。结果 （１）ＡＰＣ基因阳笥率以正常胃黏膜最高,为８３．３％,轻、中、重度异型增生分别为７７．８％、６２．５％和２５．９％,重度者明显低于前两者,癌组织则更低,为６．７％～８．０％。（２）肠上皮化生中,ＡＰＣ基因阳性率大肠型高于小肠型,水完全性高于完全性,大肠型不完全性最高。（３     

p16和CDK4在慢性萎缩性胃炎中的表达及其意义

为探讨ｐ１６和ＣＤＫ４在慢性萎缩性胃炎的组织病理学分型中的表达及其意义，对９２例慢性萎缩性胃炎的活检胃粘膜标本进行组织形态观察，以正常胃粘膜和胃腺癌作对照，采用Ｓ－Ｐ免疫组化方法，观察ｐ１６和ＣＤＫ４基因表达，结果ｐ１６在正常胃粘膜和ＣＡＧ的单纯型、增生型、肠化型中均有较高的表达，ＣＤＫ４则多不表达或弱阳性表达，而ＣＡＧ的异型性型中ｐ１６阳性表达明显减少，ＣＤＫ４阳性表达明显增多，ＣＡＧ的异型性型与管状腺癌之间无显著性差异（Ｐ＞０．０５）。结果表明ｐ１６与ＣＤＫ４在胃粘膜良恶性病变中有相反的阳性表达，但在胃癌前病变中有相关关系，两者联合检测有助于胃癌前病变的诊断；ＣＡＧ的异型性型是重要的癌前病变     

食管癌前病变组织p16及FHIT基因甲基化探讨

目的:探讨食管癌前病变组织p16及FHIT基因启动子区CpG岛的甲基化状况.方法:采用甲基化特异性聚合酶链反应(MSP)方法,对食管癌前病变不同阶段及鳞状细胞原位癌的病变组织进行了甲基化检测,并与同一个体相应的病变旁组织及慢性食管炎和浸润性鳞癌组织的甲基化情况进行了时比分析研究.结果:轻度、中重度不典型增生、鳞状细胞原位癌和浸润癌共95例病变组织中p16基因的甲基化频率分别为22.73%、59.09%、78.57%、64.86%:FHIT基因的甲基化频率分别为22.73%、45.45%、64.29%、67.57%.同一个体相应的95例病变旁对照组织中16例未检测成功,余79例中5例(6.33%)p16基因甲基化,3例(3.80%)FHIT基因甲基化,与病变组织相比,均有统计学差异.10例慢性食管炎组织中1例p16基因甲基化,未发现FHIT基因甲基化.结论:p16及FHIT基因甲基化在癌前病变阶段就已经存在,可能是食管癌发生的早期事件之一.     

Methylation of p16 CpG islands associated with malignant transformation of gastric dysplasia in a population-based study.

PURPOSE: Inactivation of p16 by aberrant methylation of CpG islands is a frequent event in carcinomas and precancerous lesions of various organs, including the stomach. The aim of this study is to investigate the relationship between p16 methylation and malignant transformation of human gastric dysplasia (DYS) based on follow-up endoscopic screening in a high-risk population. EXPERIMENTAL DESIGN: Genomic DNA samples were extracted from paraffin blocks of gastric mucosal biopsies that were histopathologically diagnosed as low-grade DYS from patients who developed gastric carcinomas [GCs (n = 21)] and those that did not do so (n = 21) during 5 years of follow-up. The methylation status of p16 CpG islands of each sample was detected by methylation-specific PCR, denatured high-performance liquid chromatography, and sequencing. RESULTS: Aberrant p16 methylation was observed in 5 of 21 samples of DYS that progressed to GC but in 0 of 21 samples that did not progress to GC (P = 0.048, two-sided). Sequencing results confirmed that all CpG sites were methylated in the analyzed sequence from these five p16-methylated cases. Furthermore, p16 methylation was also observed in the five subsequent GCs. Unmethylated p16 CpG islands were detected in all of the samples without p16 methylation. CONCLUSIONS: Our findings suggest p16 methylation is correlated with the malignant transformation of gastric DYS, and p16 methylation might be a useful biomarker for prediction of malignant potential of gastric DYS.     

Field methylation silencing of the protocadherin 10 gene in cervical carcinogenesis as a potential specific diagnostic test from cervical scrapings

PCDH10 is a member of the protocadherin cell adhesion molecule family, which are frequently downregulated in cancers. This study aimed to characterize the methylation silencing of the PCDH10 gene in the full spectrum of cervical carcinogenesis and to clarify if a field effect of methylation might be a target for a diagnostic test from cervical scrapings. Methylation silencing of PCDH10 was found in four of five cervical cancers and one of two cervical precancerous cell lines, which could be reversed by demethylation treatment. The same methylation was detected in 85.7% (24/28) of invasive cancer tissues, 36.4% (4/11) of high-grade squamous intraepithelial lesions, 20% (1/5) of low-grade squamous intraepithelial lesions, and none (0/17) of the normal cervical tissues from non-cancer subjects. In addition, methylation was also frequently found in histologically 'normal' cervical tissues adjacent to cancer lesions (7/13, 53.8%) and, less frequently, in vaginal and endometrial tissues (1/8, 12.5%). Further investigation of cervical scrapings revealed cancer-specific methylation of PCDH10 with a methylation rate of 71% (22/31) in invasive cancer, 27.9% (12/43) in carcinoma in situ , and none in high-grade squamous intraepithelial lesions excluding carcinoma in situ ( n  = 12), low-grade squamous intraepithelial lesions ( n  = 27), and normal controls ( n  = 66) ( P  < 10⁻¹⁶). Compared to the high-risk human papilloma virus test, PCDH10 methylation testing of cervical scrapings was more specific (92 vs 60%) but less sensitive (71 vs 96%) in detecting invasive cervical cancer. This study demonstrated field methylation of the PCDH10 gene specifically in the invasion stage of cervical carcinogenesis, which might be used to develop a highly specific diagnostic test for cervical scrapings. ( Cancer Sci 2009)     

Quantitative analysis of cathepsin L mRNA and protein expression during oral cancer progression

Although an important risk factor for oral cancer is the presence of epithelial dysplasia, most of these lesions will not progress to malignancy. Presently, for the individual patient with dysplasia, there are few reliable markers that may indicate the likelihood of progression to oral cancer. Cathepsin L is a lysosomal protease that degrades extracellular matrix material. Because cathepsin L is frequently overexpressed in oral squamous cell carcinoma (SCC) we hypothesized that it is also overexpressed in oral premalignancy and that premalignant lesions that progressed to oral cancer expressed higher levels of cathepsin L than those premalignant lesions that did not. In this retrospective pilot study we examined changes in cathepsin L expression at the mRNA level using quantitative TaqMan RT-PCR and at the protein level by immunohistochemistry in 33 routinely processed oral dysplastic lesions and 14 SCCs obtained from 33 patients. Sixteen of the dysplastic lesions progressed to oral SCC and 17 did not after several years of follow-up. Cathepsin L mRNA was overexpressed in 16/33 (48%) dysplastic lesions and in 9/14 (64%) oral SCC. Cathepsin L protein was also overexpressed in a large proportion of dysplasias and cancers. Overexpression was independent of dysplasia grade and identified in both those patients who progressed to oral SCC and in those who did not. Levels of cathepsin L mRNA and protein did not differ significantly in the progressing versus non-progressing dysplasias (P=0.27). However, cathepsin L mRNA and protein were significantly lower in the non-progressing dysplasias when compared to the oral cancers (P=0.03) but not in the progressing dysplasias suggesting a trend for dysplasias with overexpressed cathepsin L to be more likely to progress to oral cancer.     

Tissue carcinoembryonic antigen and DNA aneuploidy in precancerous and cancerous colorectal lesions

Chronic inflammatory bowel disease (CIBD) and colorectal adenoma are considered as precancerous conditions and lesions of large bowel carcinoma, respectively. They, therefore, may be used to study the behavior of such different factors as tumor-associated antigens and nuclear DNA content abnormalities in colorectal carcinogenesis. Tissue concentrations of carcinoembryonic antigen (CEA) were significantly higher in those precancerous lesions (CIBD: 61 +/- 11.2 ng/mg, adenoma: 70 +/- 6 ng/mg; mean +/- standard error of the mean) than in normal colonic mucosa (36 +/- 4.7 ng/mg). Colorectal carcinoma had still higher tissue levels (437 +/- 108.2 ng/mg). No correlation between tissue CEA and tumor differentiation could be found, but there was a significant difference between aneuploid (747 +/- 354 ng/mg) and diploid (139 +/- 43 ng/mg) tumors. Using flow cytometry DNA aneuploidy was present in 31.6%, 10.5%, and 51.6% of CIBD, colorectal adenoma, and carcinoma, respectively. These data suggest that the occurrence of aneuploidy is not strongly dependent on a malignant transformation, but it may also be present in premalignant colorectal lesions without cellular dysplasia.     

Genomic alterations in malignant transformation of Barrett's esophagus

The incidence of adenocarcinoma in Barrett's esophagus has been increasing rapidly over the past decades. Neoplastic progression is characterized by three well-defined premalignant stages: metaplasia, low-grade dysplasia, and high-grade dysplasia. A genome-wide overview, based on comparative genomic hybridization, was performed, evaluating 30 Barrett's adenocarcinomas and 25 adjacent precursors, i.e., 6 metaplasias, 9 low-grade dysplasias, and 10 high-grade dysplasias. The frequency of losses and gains significantly increased in the subsequent stages of malignant transformation. Losses of 5q21-q23, 9p21, 17p12-13.1, 18q21, and Y were revealed in low-grade dysplasias. This was followed by loss of 7q33-q35 and gains of 7p12-p15, 7q21-q22, and 17q21 in high-grade dysplasias along with high-level amplification (HLA) of 7q21 and 17q21. In the invasive cancers, additional losses of 3p14-p21, 4p, 4q, 8p21, 13q14-q31, 14q24.3-q31, 16q21-q22, and 22q as well as gains of 3q25-q27, 8q23-24.1, 12p11.2-12, 15q22-q24, and 20q11.2-q13.1 were distinguished along with HLAs of 8p12-p22 and 20q11.2-q13.1. Approximately one-third of the alterations in the dysplasias were also found in the adjacent adenocarcinomas, illustrating that multiple clonal lineages can be present in Barrett's esophagus. Novel findings include loss on 7q, gain on 12p, and the observation of several HLAs in high-grade dysplasias. Furthermore, loss of 7q33-q35 was found to represent a significant distinction between low-grade and high-grade dysplasia (P = 0.01), whereas loss of 16q21-q22 and gain of 20q11.2-q13.1 were disclosed to significantly discriminate between high-grade dysplasia and adenocarcinoma (P = 0.02 and P = 0.03, respectively). This inventory of genetic aberrations increases our understanding of malignant transformation in Barrett's esophagus and might provide useful biomarkers for disease progression.     

MDM2/p53 co-expression in oral premalignant and malignant lesions: potential prognostic implications.

The expression of MDM2 protein in betel and tobacco related oral malignancies in Indian population, its relationship to clinicopathological parameters and p53 protein expression was investigated. Sixty five oral squamous cell carcinomas (SCCs), 33 premalignant lesions (leukoplakia) and 30 normal oral tissues were assessed by immunohistochemical analysis. MDM2 protein was overexpressed in 51/65 (78%) oral SCCs and 17/33 (52%) premalignant lesions; 11/23 hyperplastic lesions and 6/10 dysplastic lesions. mdm2 gene amplification is an infrequent event in oral tumorigenesis. Elevation in the level of MDM2 protein not only in oral SCCs but also in premalignant lesions suggests that altered MDM2 expression is an early even in the pathogenesis of oral neoplasia. The hallmark of the study was the significant association of MDM2 expression with the p53 protein accumulation in 16/33 (49%) oral premalignant lesions (p = 0.001) and 39/65 (60%) malignant lesions (p = 0.021), suggesting an active role for MDM2 in binding and inactivating p53 in oral tumorigenesis. Further, significant association of MDM2/p53 co-expression was observed with advanced tumour stage (p = 0.0009), as well as lymph node metastasis (p = 0.0325) features associated with aggressive tumour behaviour and poor prognosis. Discordant MDM2+/p53-phenotype was observed in 12/65 (18%) oral SCCs suggesting a p53-independent role for MDM2 in the pathogenesis of a subset of oral carcinomas. In conclusion, alterations in MDM2 and p53 expression are early events likely to be involved in preinvasive stages in oral tumorigenesis and may be indicative of a 'gain of function' phenotype with more aggressive characteristics.     

Aurora A overexpression induces cellular senescence in mammary gland hyperplastic tumors developed in p53-deficient mice

Aurora A mitotic kinase is frequently overexpressed in various human cancers and is widely considered to be an oncoprotein. However, the cellular contexts in which Aurora A induces malignancy in vivo are still unclear. We previously reported a mouse model in which overexpression of human Aurora A in the mammary gland leads to small hyperplastic changes but not malignancy because of the induction of p53-dependent apoptosis. To study the additional factors required for Aurora A-associated tumorigenesis, we generated a new Aurora A overexpression mouse model that lacks p53. We present evidence here that Aurora A overexpression in primary mouse embryonic fibroblasts (MEFs) that lack p53 overrides postmitotic checkpoint and leads to the formation of multinucleated polyploid cells. Induction of Aurora A overexpression in the mammary glands of p53-deficient mice resulted in development of precancerous lesions that were histologically similar to atypical ductal hyperplasia in human mammary tissue and showed increased cellular senescence and p16 expression. We further observed DNA damage in p53-deficient primary MEFs after Aurora A overexpression. Our results suggest that Aurora A overexpression in mammary glands is insufficient for the development of malignant tumors in p53-deficient mice because of the induction of cellular senescence. Both p53 and p16 are critical in preventing mammary gland tumorigenesis in the Aurora A overexpression mouse model.     

Promoter methylation of p16 associated with Helicobacter pylori infection in precancerous gastric lesions: a population-based study

To investigate the relationship between p16 methylation and Helicobacter pylori infection in precancerous gastric lesions, a population-based study was conducted in Linqu County, a high-risk area of gastric cancer in China. Methylation status of p16 was evaluated by methylation-specific polymerase chain reaction in 920 subjects with precancerous gastric lesions. H. pylori status was determined by 13C-urea breath test and the density of H. pylori in biopsy specimens used for detecting methylation status was assessed by the modified Giemsa stain. The frequency of p16 methylation was significantly higher in subjects with H. pylori positive than those with H. pylori negative in each category of gastric lesion (p<0.001, respectively). Compared with H. pylori negative, the odds ratios (ORs) of p16 methylation were markedly elevated in subjects with H. pylori positive for superficial gastritis (OR, 9.45; 95% confidence interval [CI]: 2.94-30.41), chronic atrophic gastritis (OR, 15.92; 95%CI: 7.60-33.36), intestinal metaplasia (OR, 4.46; 95%CI: 2.44-8.13), indefinite dysplasia (OR, 3.67; 95%CI: 1.90-7.10), and dysplasia (OR, 2.48; 95%CI: 1.02-5.99). Moreover, the frequencies of p16 methylation increased steadily with the severity of H. pylori density in gastric mucosa. Compared with H. pylori negative, the OR of p16 methylation was 1.02-16.13 times higher in subjects with mild H. pylori infection, and 2.69-38.73 times higher in those with moderate/severe infection, respectively. Our findings indicate that p16 methylation was significantly associated with H. pylori infection in precancerous gastric lesions, suggesting that H. pylori infection could potently induce methylation of p16 CpG island.     

Expression of the p16(INK4a) gene product, methylation of the p16(INK4a) promoter region and expression of the polycomb-group gene BMI-1 in squamous cell lung carcinoma and premalignant endobronchial lesions

It is generally assumed that squamous cell carcinoma develops in a stepwise manner from normal bronchial epithelium towards cancer by the accumulation of (epi)genetic alterations. Several mechanisms including mutations and homozygous deletions or hypermethylation of the p16(INK4a) promoter region can cause loss of p16 expression. Recent studies suggest overexpression of the polycomb-group gene BMI-1 might also down-regulate p16 expression. In this study, we analyzed the p16 expression in relation to the methylation status of the p16 promoter region of the p16(INK4a) gene and the expression of BMI-1 in bronchial squamous cell carcinomas (SCC) and its premalignant lesions. Nine (69%) SCC showed loss of p16 expression and 10 (77%) showed expression of BMI-1. Of four p16 positive samples two (50%) were BMI-1 positive, whereas among nine p16 negative samples, eight (89%) revealed BMI-1 staining. Four (44%) p16 negative samples were hypermethylated at the p16(INK4a) promoter region; the other p16 negative tumors that showed no hypermethylation revealed BMI-1 staining. Only two premalignant lesions showed absence of p16 expression, of which one (carcinoma in situ) was hypermethylated at the p16(INK4a) promoter region and the other (severe dysplasia) showed BMI-1 expression. In total, 11 precursor lesions (48%) revealed BMI-1 expression. In conclusion, the results of this study suggest that loss of p16 expression by promoter hypermethylation is inconsistently and occurs late in the carcinogenic process at the level of severe dysplasia. To what extent overexpression of the polycomb-group protein BMI-1 attributes to down regulating of p16 expression remains unclear.     

Increased protein expression of DNA methyltransferase (DNMT) 1 is significantly correlated with the malignant potential and poor prognosis of human hepatocellular carcinomas

Alteration of DNA methylation is one of the most consistent epigenetic changes in human cancers. DNA methyltransferase (DNMT) 1 is a major enzyme involved in establishing genomic methylation patterns. Most of the studies concerning DNMT1 expression in human cancers have been performed only at the mRNA level. To directly examine DNMT1 protein expression levels during human hepatocarcinogenesis, 16 histologically normal liver tissues, 51 noncancerous liver tissues exhibiting chronic hepatitis or cirrhosis, which are considered to be precancerous conditions, and 53 hepatocellular carcinomas (HCCs) were subjected to immunohistochemic examination. If more than 20% of the cells exhibited nuclear DNMT1 staining, the tissue sample was considered to be DNMT1-positive. DNMT1 immunoreactivity was observed in 23 (43%) of the HCCs, but in none (0%) of the histologically normal liver or noncancerous liver tissues exhibiting chronic hepatitis or cirrhosis. The incidence of increased DNMT1 protein expression in HCCs correlated significantly with poor tumor differentiation (p = 0.0006) and portal vein involvement (p = 0.0002). Moreover, the recurrence-free (p = 0.0001) and overall (p < 0.0001) survival rates of patients with HCCs exhibiting increased DNMT1 protein expression were significantly lower than those of patients with HCCs that did not exhibit increased expression. Increased DNMT1 protein expression may play a critical role in the malignant progression of HCCs and be a biologic predictor of both HCC recurrence and a poor prognosis in HCC patients.     

p53 overexpression in oral lichen planus

Oral lichen planus (OLP) is considered as a premalignant lesion, but some argue that only lichenoid lesions with dysplasia is precancerous. To address the question whether OLP without dysplasia is premalignant, we investigated the immunohistochemical expression of p53 in OLPs without dysplasia. Half of the OLPs showed p53 positive cells in the basal epithelium. SSCP-PCR analysis of 4 p53 positive OLPs failed to demonstrate mutations. What is the significance of p53 expression in these OLPs? The confinement of p53 positive cells to the basal cells seems to be against false positivity. Since the pathogenesis of OLPs involves cell-mediated cytotoxicity (CMC) which causes marked apoptosis, it is possible that the p53 expression represents wild-type p53 that may be regulating the apoptosis. Alternatively p53 protein may be stabilized by some mechanisms other than gene mutation as a result of cellular insults from CMC.     

Immunohistochemically stained markers (p53, PCNA, bcl-2) in dysplastic lesions of the larynx.

The percentage of malignant transformation of laryngeal dysplastic lesions is difficult to estimate. There is a need for new histological markers which could enable more objective assessment of the premalignant stages of the larynx and help in estimation of the potential of future neoplastic progression. We performed a retrospective study to determine whether immunohistochemical staining for the proliferating cell nuclear antigen (PCNA), tumour suppressor gene protein p53 and antiapoptotic protein bcl-2 may be prognostic factors in laryngeal epithelial lesions. Staining was performed on 57 paraffin-embedded biopsies from patients with clinically detected precancerous stages of the larynx. Histopathologic examination revealed normal epithelium in six cases, mild dysplasia in 20 cases, moderate dysplasia in 18 cases, severe dysplasia in seven cases, CA in situ in four cases, papilloma in one case and CA invasivum in one case. The p53 count in mild and moderate dysplasia was 26.8 and 38.6%, respectively. This difference was statistically significant. There was significant correlation between PCNA and p53 scores. There was also a relationship between the scores of these markers and bcl-2 expression. In ten out of 45 cases of dysplastic lesions the invasive cancer developed in 4 years of follow-up. The correlation between PCNA score and malignant progression of the dysplastic lesions was on the statistical borderline. There was significant relationship between malignant transformation and age of the patients.     

MTA1和TSLC1表达与结肠腺瘤癌变相关性研究

目的 结肠腺瘤是结肠癌的癌前病变,积极诊断和治疗结肠腺瘤是控制、减少结肠癌发病的重要途径.本研究探讨肿瘤转移相关基因1(metastsis associated gene 1,MTA1)和肺癌肿瘤抑制因子1(tumor suppressor in lung cancer 1,TSLC1)在结肠腺瘤和结肠癌组织中的表达,以及MTA1和TSLC1在结肠腺瘤癌变过程中的作用.方法 选择2010-04-01-2015-12-31河北医科大学附属邢台市人民医院手术切除的结肠癌标本104例、肠镜活检结肠腺瘤标本114例及癌旁正常结肠组织(距离癌组织>3 cm)30例为研究对象,应用免疫组织化学法检测3组标本中MTA1和TSLC1蛋白的表达.结果 正常结肠组织中MTA1阳性表达率为6.67%,管状腺瘤为42.86%,绒毛状腺瘤为63.64%,结肠癌为84.62%,呈逐渐上升趋势,χ2=68.668,P<0.001;中重度异型增生结肠腺瘤中MTA1的阳性表达率(81.25%)高于轻度异型增生结肠腺瘤(39.02%),χ2=16.421,P<0.001;正常结肠组织中TSLC1阳性表达率为96.67%,管状腺瘤为88.57%,绒毛状腺瘤为63.64%,结肠癌组为42.31%,呈逐渐下降趋势,χ2=52.679,P<0.001;中重度异型增生结肠腺瘤中TSLC1阳性表达率(37.50%)明显低于轻度异型增生结肠腺瘤(95.12%),差异有统计学意义,χ2=45.982,P<0.001.结论 结肠癌组织中MTA1呈高表达,TSLC1表达缺失.MTA1和TSLC1参与正常组织、癌前病变和癌组织的发生发展,在结肠腺瘤的癌变过程中起着重要的作用.