Fibroblast control on epithelial differentiation is gradually lost during in vitro tumor progression

This study aimed to investigate the role of underlying fibroblasts on morphogenesis of in vitro epithelium reconstituted with normal and neoplastic human oral keratinocytes at various stages of malignant transformation. Primary normal human oral keratinocytes (NOKs), early neoplastic/dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ 15 cell line) were organotypically grown on top of a collagen type I matrix with or without primary normal human oral fibroblasts. Morphogenesis of the reconstituted epithelia was assessed by histomorphometry, immunohistochemistry (Ki-67, cyclin D1, cytokeratin 13 (CK13), collagen IV, E-cadherin, p53, CD40), and the terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end-labelling method. Reproducible in vitro models of multistage oral carcinogenesis were established. Presence of fibroblasts in the collagen matrix significantly increased cell proliferation in all three models (p<0.05), and induced an invasive pattern of growth in the neoplastic cell lines (p<0.05). In normal, but not in neoplastic oral keratinocytes fibroblasts induced the expression of CD40, and polarized the expression of E-cadherin and p53 to the basal cell layer. In both normal and early neoplastic keratinocytes (DOK cell line), fibroblasts induced the expression of CK13 and collagen IV. In the neoplastic oral keratinocytes (PE/CA-PJ 15 cell line), the presence of underlying fibroblasts did not change the expression of any of the protein markers assessed. This study showed that (1) major steps of oral carcinogenesis can be reproduced in vitro, and (2) the tight control exerted by fibroblasts on epithelial morphogenesis of in vitro reconstituted normal human oral mucosa is gradually lost during neoplastic progression.     

Diagnostic value of nucleolar organizer regions (AgNORs) in brush biopsies of suspicious lesions of the oral cavity.

OBJECTIVE: The aim of this retrospective study was to report on the diagnostic accuracy of AgNOR-analysis as an adjunctive diagnostic tool of conventional oral exfoliative cytology taken from suspicious lesions in our clinic. STUDY DESIGN: Cytological diagnoses obtained from brush biopsies of macroscopically suspicious lesions of the oral mucosa from 75 patients (final diagnoses: 53 histologically proven squamous cell carcinomas, 11 leukoplakias and other inflammatory oral lesions) and from 11 patients with normal mucosa as a negative control group were compared with histological and/or clinical follow-ups. Five smears were doubtful and seven suspicious for tumor cells in the cytologic report. Number of AgNOR's were counted in 100 squamous epithelial cell-nuclei per slide after silver-restaining. RESULTS: Sensitivity of our cytological diagnosis alone on oral smears for the detection of squamous carcinomas was 92.5%, specificity 100%, positive predictive value was 100% and negative 84.6%. The best cut-off value of the mean number of AgNOR dots per nucleus distinguishing benign from malignant cells was 4.8. The percentage of nuclei with more than three AgNORs had a cut-off level of 70%. Applying these methods to twelve doubtful or suspicious cytological diagnoses we were able to correctly establish the diagnosis of malignancy in ten cases of histologically proven cancers and to reveal benignity in two histologically proven cases. Thus we achieved a positive and negative predictive value of 100% each. CONCLUSIONS: Smears from brushings of visible oral lesions, if clinically considered as suspicious for cancer, are an easily practicable, non-invasive, painless, safe and accurate screening method for detection of oral cancerous lesions. We conclude that AgNOR-analysis may be a useful adjunct to other methods in routine cytological diagnosis of oral cancer that can help to solve cytologically suspicious or doubtful cases.     

PCR based detection of HPV 16 and 18 genotypes in normal oral mucosa of tobacco users and non-users

There is increasing evidence of a causal association between human papillomavirus (HPV) and oral squamous cell carcinoma (OSCC). Several studies have shown that HPV is associated with increased risk of oral cancer independent of exposure to tobacco and alcohol. The association is valid for HPVs 16 and 18, which generally are considered high risk types, because they have been detected in oral dysplastic lesions and cancers. We determined the baseline prevalence of HPVs 16 and 18 in normal oral mucosa of individuals with and without tobacco habit. PCR was used for DNA collected by oral smears to detect HPV 16/18 DNA in normal oral mucosa of 60 healthy individuals who were assigned to two groups of 30 subjects each. One group had a tobacco habit, the other did not. The tobacco user group comprised individuals who were tobacco chewers only. Sixty-five percent of individuals were positive for HPV 16/18 DNA, but HPV 16/18 positivity was less in individuals with tobacco habit than in those without tobacco habit. No significant association was found between the presence of HPVs and gender, age or duration of chewing habit, or between groups with and without a tobacco habit. We propose that HPVs16 and 18 commonly are present in normal oral mucosa and emphasize the importance of distinguishing clinical, subclinical and latent HPV infections when investigating HPVs and OSCC.     

Micronuclei and other nuclear anomalies in normal human buccal mucosa cells of oral cancer patients undergoing radiotherapy: a field effect

We evaluated micronuclei and other nuclear anomalies in exfoliated epithelial cells of the oral cavity on the side opposite the lesion targeted by radiotherapy and correlated them with radiation doses. Buccal smears were obtained from oral cancer patients undergoing radiotherapy with a cumulative dose of at least 1000 rad for 3 weeks and from controls matched for age, gender and habits. The exfoliated cells from the mucosa were collected using a cytobrush; smears were prepared, fixed in 80% methanol and stained using the Feulgen plus fast green method. The mean number of micronuclei and other nuclear anomalies/1000 cells was significantly greater in patients undergoing radiotherapy treatment, but the differences were not significant compared to radiation doses. It appears that radiotherapy has a potent clastogenic effect on buccal mucosal cells of oral cancer patients.     

The characteristics of protein p53 expression in the mucosal epithelium of the large intestine affected by polyps and cancer

The direct dependence between p53 expression in epithelial cells and the level of morphological malignant neoplasm was established as the result of investigation of expression of this biomolecular marker in 12 polyps and 35 colorectal cancer. It was shown that family cancer syndrome 1 type was observed in pedigrees of majority patients with p53 positive cancers.     

Comparison of Planimetry and Image Analysis For the Discrimination Between Normal and Abnormal Cells In Cytological Smears of Suspicious Lesions of the Oral Cavity

Light microscope analysis of cytological smears of suspicious lesions of the oral cavity is used as a method for detecting early cancer in the oral cavity. the sensitivity of this approach can be improved by quantitative analysis of the cells in the cytological smears. We have compared the efficiency of planimetry and the Vids V system of image analysis, as quantitative methods for discriminating between normal and abnormal cells in cytological smears of suspicious lesions in the oral cavity. Both methods detected an increase in nuclear area and a decrease in cytoplasmic area in abnormal epithelial cells from dysplastic lesions of increasing severity. However, image analysis was better able to discriminate between benign and malignant cells on the basis of nuclear size. Thus the Vids V system of image analysis is more appropriate than planimetry for quantitative analysis of cytological smears from the oral cavity.     

Sensitivity of Seescan TV image analysis in discriminating between normal, dysplastic and malignant oral smears

OBJECTIVE: Smears from premalignant and malignant lesions may contain large proportions of normal cells together with atypical cells; that could reduce the sensitivity of cytologic diagnosis. The present study assessed the performance of the Seescan TV image analysis system (TVIAS) in distinguishing between normal, premalignant and malignant oral smears. The sensitivity of Seescan TVIAS was tested using both white and monochromatic light. STUDY DESIGN: Nuclear area (NA) and corrected integrated optical density (IOD) of 50 Feulgen-stained nuclei were measured in smears collected from normal oral mucosa (n = 6), lesions displaying epithelial dysplasia (n = 5) and invasive squamous cell carcinoma (n = 5) using a Seescan TVIAS with both white and monochromatic light. RESULTS: There was a significant increase (P < .001) in mean IOD for nuclei in smears from dysplastic lesions and carcinomas as compared with normal smears. For smears from carcinomas, the mean NA was significantly elevated as compared with dysplastic (P < .001) and normal smears (P < .01). Mean NA for dysplastic smears was significantly reduced as compared with normal smears. While all smears from premalignant and malignant lesions contained mostly normal nuclei, a significant proportion of abnormal nuclei was identified in each smear. CONCLUSION: Although oral smears contain large amounts of normal cells, the Seescan TVIAS could successfully identify dysplastic and malignant cells on the basis of both IOD and NA values with or without the use of a monochromatic filter.     

p27和p53基因在大肠癌中的表达及临床意义

目的 研究大肠癌患者癌组织中p27、p53基因的表达及其相互之间的关系,以探讨p27、p53基因在大肠癌发生中的作用及临床意义。方法 运用原位杂交方法及免疫组化SP法检测58例大肠癌组织及正常黏膜中p27mRNA和P27蛋白的表达,同时运用免疫组化法分析相同组织中P53蛋白表达状况。结果 p27mRNA在大肠癌组织及正常黏膜中的表达阳性率均为100%。P27蛋白在大肠癌组织中的表达阳性率为55.17%,在正常黏膜中的表达阳性率为96.55%（P〈0.01）;癌组织中P53蛋白表达阳性率为53.45%,正常黏膜未见P53蛋白表达（P〈0.01）;大肠癌组织中P27与P53蛋白表达无明显相关性。P27蛋白的表达与肿瘤分化程度呈负相关（P〈0.01）,与临床其它病理因素均无相关性（P〉0.05）。大肠癌组织中P53蛋白表达与临床病理因素亦无相关性（P〉0.05）。结论 P27蛋白表达的调控主要在转录后水平,P27蛋白检测可作为评价大肠癌恶性程度和预后判断的重要指标。P27及P53蛋白在大肠癌的发生发展过程中具有重要作用。     

p53--prognostic factor of malignant transformation of Barrett's esophagus

The most significant precancerosis in the esophageal cancer is Barrett's esophagus. The risk of malignant transformation is determined primarily in accordance with the degree of dysplastic alterations of the mucosa. Indication of "preventive" extirpation of the esophagus should be supported by other factors, for example by detection of p53 mutation or expression. The study reports on the evaluation of a group of 20 patients with Barrett's esophagus treated at the 1st Department of Surgery, the p53 level and its correlation with histological findings evaluated in these patients. A good correlation was found between the grade of Barrett's esophagus dysplasia and high p53 positivity. This correlation was also confirmed by detection of early carcinoma in patients with "preventive" extirpation of the esophagus due to a high-grade dysplasia. Preliminary results show that examination of p53 level in specimens taken from the esophageal mucosa may be helpful for the estimation of malignant potential of the dysplastic mucosa.     

p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.     

Analysis of silver binding nucleolar organizer regions in exfoliative cytology smears of potentially malignant and malignant oral lesions

Nucleolar organizer regions are nucleolar components that contain proteins that are stained selectively by silver methods; they can be identified as black dots throughout the nucleolus and are known as silver binding nucleolar organizer regions (AgNOR). The number of AgNOR is related to the cell cycle and the proliferative activity of the cells. We investigated AgNOR using exfoliative cytology smears of potentially malignant oral lesions. Eighty individuals were divided into four equal groups: healthy controls, oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. The mean number of AgNOR in each study group gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. The proliferative index was increased in the oral premalignant and malignant patients compared to normal subjects. The mean AgNOR size gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. Spherical shaped AgNOR were most common in controls, whereas large, clustered and kidney shapes were most common in oral squamous cell carcinoma. Multiparameter analysis of AgNOR in oral exfoliative smears is a simple, sensitive and cost-effective method for differentiating premalignant from malignant lesions and can be used in conjunction with routine cytomorphological evaluation.     

Quantitative cytomorphologic analyses of Papanicolaou-stained smears from oral lichen planus

OBJECTIVE: To apply quantitative techniques to cytologic smears in order to improve the diagnostic sensitivity of cytology in the detection of malignant change in the oral cavity. STUDY DESIGN: Eighty-two patients with lesions of oral lichen planus were investigated. A total of 247 Papanicolaou-stained buccal smears underwent nuclear and cytoplasmic area measurements using a TV image analyzer. RESULTS: The cytomorphologic results of this study suggest that lesions of oral lichen planus contain smaller cells and nuclei than those observed in clinically normal oral mucosal smears. There was a statistically significant reduction in nuclear area (P < .001) and cytoplasmic area (P < .05) for the oral lichen planus smears. CONCLUSION: The TV image analysis system could provide a valuable method of quantifying cell changes in oral smears collected from oral mucosa as a method of monitoring lesions of oral lichen planus.     

口腔白斑组织癌变标志性基因筛查

口腔黏膜白斑病（oral leukoplakia,OLK）是最常见的口腔粘膜的癌前病变．OLK恶性转化率高达43%,因此加强其风险调查和监测对于该病的预防和治疗具有重要意义．本研究采用美国SuperArray公司的肿瘤基因芯片（OHS-802）检测口腔正常组织、OLK组织和口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC)组织的肿瘤相关基因表达差异,旨在筛查与口腔黏膜白斑病发生和癌变相关的表达阳性基因,为探究OLK的发生及癌变机制奠定基础．将“正常组织→OLK组织→OSCC组织”连续递增2倍以上或递减2倍以上的基因确定为OLK组织癌变的标志性基因．SuperArray基因芯片、RT-PCR法和实时定量PCR结果显示,仅有3个基因（ACP-2、BCL-2、SOCS-3）表达水平连续下调2倍以上,有6个基因（CLK-3、CTNNB-1、FKBP-8、GDF-15、NF-1、XRCC-1）表达连续上调2倍以上．进一步经RT-PCR和实时定量PCR验证,其结果与SuperArray结果一致．本研究结果提示,ACP-2等9个基因可能是口腔正常黏膜组织经OLK转变为癌组织的驱动基因,可作为OLK组织、OSCC组织和口腔正常黏膜组织诊断的分子标志．     

Comparative study of the expression of Rb and p53 genes in human colorectal cancers,colon carcinoma cell lines and synchronized human fibroblasts

We have compared the expression of the retinoblastoma (Rb) and p53 genes in normal human fibroblasts, colon carcinoma cell lines, matched pairs of colorectal tumor tissues and adjacent normal mucosa and in synchronized human diploid fibroblast cell line W138. The increased expression of Rb and p53 RNA was observed in a majority of colorectal cancers in comparison to adjacent normal mucosa and is accompanied by proportional increase in the expression of histone H3 gene. The Rb and p53 RNA levels varied significantly between the various colon carcinoma cell lines. However, we found that the expression of Rb and p53 RNA is regulated differently in cell cycle synchronized normal human fibroblasts. The Rb mRNA level did not change with the position in the cell cycle and did not differ significantly whether the cells were serum deprived or in 10% serum. But p53 mRNA expression follows the same pattern as histone H3 mRNA.     

Image analysis of p53 and cyclin D1 expression in premalignant lesions of the oral mucosa

OBJECTIVE: The expression of p53 and cyclin D1 proteins was analyzed by image analysis in oral premalignant lesions and normal oral mucosa. STUDY DESIGN: Punch biopsies from the normal oral mucosa were obtained from 20 normal donors and 41 patients with oral dysplastic leukoplakias. After controlled formaldehyde fixation and paraffin embedding, immunohistochemistry was used to detect cyclin D1 and p53. Image analysis was performed using stain intensity levels established by determining color thresholds (nuclear score) in the basal and parabasal layers. RESULTS: Analysis of sections showed a similar pattern: only two normal donors had a few intensely positive p53 cells in the basal layer of the floor of the mouth and the tongue epithelia. Similarly, only three donors had intensely positive cyclin D1 cells in the normal epithelia of the same sites. Most cells fell in the range of negative or marginal stain (lower quartiles or terciles of nuclear score). These data on normal mucosa were compared with low grade oral leukoplakias (LGD) with mild to moderate dysplasia and with high grade leukoplakias (HGD) with severe dysplasia. Both markers were differentially expressed in precursor lesions versus normal epithelia. Statistical analysis of our data shows that the intensity of the immunohistochemical stain, as reflected in the nuclear scores of p53, is a reliable parameter that can differentiate between LGD and HGD of the oral mucosa. This was especially true when higher nuclear scores were compared. In contrast, low nuclear scores are more effective in differentiating normal epithelia from dysplastic epithelia. Although cyclin D1 immunohistochemistry does not stain as intensely as p53 stain, similar conclusions can be derived from those data. CONCLUSION: Image analysis of these two markers proved useful in distinguishing normal oral epithelia from low grade and high grade leukoplakias. With further developments in this field it is hoped that image analysis procedures could be used in different types of studies in which variations of protein expression in tissue sections could have prognostic implications or could be useful to determine subtle effects of curative or preventive treatment.     

p53 Codon 72 Polymorphism as a Risk Factor in the Development of HPV-Associated Cervical Cancer

Human papilloma virus (HPV) has been implicated in cervical carcinoma, and the p53 gene is polymorphic at amino acid 72 of the protein that it encodes. The association between p53 polymorphisms and risk for HPV-associated cervical cancer has been examined, but the results have been conflicting. It has been reported that patients with the arginine form have a higher risk of developing cervical cancer than those with the proline form. The purpose of this study was to examine whether p53 Arg at the polymorphic position 72 could represent a risk factor for women with high-risk HPV-associated premalignant and malignant cervical lesions. The study was carried out on 60 smears from patients with high-risk HPV-related cervical lesions. Also, 74 HPV-negative normal smears and 61 normal blood samples were used as controls. HPV-18 was the most frequent type. There was a difference in the distribution of p53 genotypes between high-risk HPV-cervical lesions and the normal samples. The allele frequency of p53 Arg/Arg was much higher than the normal samples (46.5% versus 20.5% in HPV-negative normal smears and 20% in blood samples). Based on the findings of this study, it is suggested that p53 Arg homozygosity could possibly represent a potential risk factor for the tumorigenesis of the cervix. Statistically significant correlation was not observed between the presence of Arg/Pro homozygosity or Arg/Pro heterozygosity and HPV typing.     

Overexpression and clinicopathological significance of homeobox gene Quox-1 in oral squamous cell carcinoma

The expression and clinicopathological significance of Quox-1 gene was studied in oral squamous cell carcinoma (OSCC). Immunocytochemistry and western blot analysis were used to examine the different expressions of Quox-1 protein in 114 OSCC specimens, 34 oral epithelial dysplasia specimens, and 16 normal oral mucosa specimens. RT-PCR and virtual Northern Blot were also used to examine the expression of Quox-1 mRNA. It was found that Quox-1 was not expressed in normal epithelium. However, as dysplastic lesions progressed Quox-1 expression increased (p < 0.01), and Quox-1 expression was not significantly different between severe dysplasia and highly differentiated OSCCs (p > 0.05). As the degree of differentiation decreased, Quox-1 positivity increased in OSCC (p < 0.01), and the rate of Quox-1 (81.58%) positivity in OSCC was higher than that in normal oral mucosa (p < 0.01). Our findings imply that the positive expression of Quox-1 is correlated with the histological classification of OSCCs. Thus, the expression of Quox-1 in OSCC may serve as a significant predicting factor of proliferative status and malignant degree, and it may also be a biological detection marker of oral mucosas initial cancer and of OSCC.     

p53突变蛋白在胃癌组织中的表达及免疫电镜观察

作者应用抗ｐ５３单克隆抗体Ｐａｂ１８０１（Ａｂ２美国癌基因公司产品）对３８例手术切除的胃癌组织及癌旁胃粘膜的冰冻切片标本进行ｐ５３突变蛋白表达的检测,并进一步用胶体全免疫电镜技术对ｐ５３突变蛋白的分布特征进行观察。结果：３８例胃癌组织中,２４例有ｐ５３突变蛋白高表达,阳性率６３．２％。在对应的癌旁胃粘膜中１０例为ｐ５３的弱表达,正常组织无表达。伴有淋巴结转移的２３例胃癌标本中,１８例ｐ５３高表达（７８．３％）。免疫电镜结果表现,ｐ５３蛋白主要分布于核内染色质中,胞浆中有散在的阳性区,但以核膜周边为主,紧靠核膜。本研究结果提承胃癌的发生及其肿瘤的生物学行为与抑癌基因ｐ５３的突变密切相关,ｐ５３突变蛋白可能是通过对ＤＮＡ复制的影响而参与肿瘤的形成。     

Quantitative cell-cycle protein expression in oral cancer assessed by computer-assisted system

The knowledge of cell-cycle control has shown that the capacity of malignant growth is acquired by the stepwise accumulation of defects in specific genes regulating cell growth. Histologic diagnosis might be improved by a quantitative evaluation of more specific diagnosis biomarkers, which could help to precisely identify pre-malignant and malignant oral lesions. The aim of the present study is to evaluate whether computer-based quantitative assessment of p53, PCNA and Ki-67 immunohistochemical expression, could be used clinically to foresee the risk of oral malignant transformation. This retrospective study was carried out in ninety-five oral biopsies, 27 were classified as fibrous inflammatory hyperplasia, 40 as leukoplakia and 28 as oral squamous cell carcinoma. Sixteen out of the 40 leukoplakia were diagnosed as non-dysplastic leukoplakia, the other 24 being dysplastic leukoplakia, of which 50.0% were classified as moderate to severe dysplasia. Comparison of the four groups of oral tissues showed significant rises in p53 and Ki-67 positivity index, which increased steadily in the order benign, pre-malignant, and malignant. In contrast, it was not possible to relate higher PCNA levels with pre-malignant and malignant oral lesions. We therefore conclude that PCNA immunohistochemistry expression is probably an inappropriate marker to identify oral carcinogenesis, whereas joint quantitative evaluation of p53 and Ki-67, appears to be useful as a tumor marker, providing a pre-diagnostic estimate of the potential for cell-cycle deregulation of the oral proliferate status.