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.     

Malignancy associated changes in epithelial cells of buccal mucosa: a potential cancer detection test

OBJECTIVE: To analyze the presence of malignancy associated changes (MACs) in normal buccal mucosa cells of lung and breast cancer patients and their relationship to tumor subtype, stage and size. STUDY DESIGN: Buccal mucosa smears of 107 lung cancer and 100 breast cancer patients and corresponding healthy subjects were collected, stained by the DNA-specific Feulgen-thionin method and scanned using an automated high-resolution cytometer. Nuclear texture features of a minimum of 500 nuclei per slide were calculated, and statistical classifiers using Gaussian models of class-probability distribution were designed, trained and tested in 3 parts: (1) ability to separate cancer patient samples from controls, (2) cross-validation of classifiers for different cancer types, and (3) correlation of MAC expression with tumor subtype, stage and size. RESULTS: Lung and breast cancer induce MACs in normal buccal mucosa cells. The classifiers based on the selected nuclear features correctly recognized >80% of lung and breast cancer cases. The results indicate that MAC detection is not dependent on the tumor subtype, stage or size. CONCLUSION: The presence of MACs in buccal mucosa cells offers the potential for developing a new noninvasive cancer screening test.     

Karyometric features in nuclei near colonic adenocarcinoma. Statistical analysis.

The normal mucosa adjacent to colonic adenocarcinoma (marginal or transitional mucosa) has been shown to have subtle alterations of architecture, surface glycoproteins and proliferative activity. To evaluate possible changes in nuclear configurations in this marginal mucosa, a large set of cytometric features was evaluated using a computer-assisted video analysis system. Preliminary statistical analysis of the measurements identified six nuclear features useful for discriminating marginal mucosa nuclei from normal (control) mucosa nuclei: total optical density (OD), nuclear area, chromatin texture (from gray value cooccurrence matrix), chromatin coarseness, average OD of nuclear staining and peripheral tendency of the chromatin in the nucleus. An analysis of variance revealed that both patient-to-patient and gland-to-gland variation would limit the usefulness of any one feature as a screening tool. As a group, however, these six features should be investigated further as markers of preneoplastic changes in histologically normal-appearing mucosa.     

Cytometric analysis of oral scrapings of patients with oral lichen planus

A. Acha‐Sagredo, Y. Jiménez, J. V. Bagán, M. A. Echebarria‐Goicouria and J. M. Aguirre‐Utric analysis of oral scrapings of patients with oral lichen planus Objective: Aneuploidy has been associated with malignant and premalignant oral lesions. In the past few years, its application in oral precancerous lesions and its prognostic meaning have been controversial issues. The aim of our study was to characterize alterations in DNA content by automated DNA image cytometry in oral scrapings of patients with oral lichen planus. Methods: Cytological samples from 40 patients clinicopathologically diagnosed with oral lichen planus were analysed by DNA image cytometry. Results: All the cases were classified as diploid, showing a predominant population of cells with normal DNA content (DNA index, 0.85–1.15). Atrophic/erosive lesions showed a higher percentage of tetraploid cells when compared with reticular/papular lesions but this was not statistically significant (P = 0.09). Conclusions: Aneuploidy does not seem a common event in oral lichen planus lesions. However, we consider that the use of DNA image cytometry of oral scrapings may be an easy and helpful methodology in the follow‐up of patients with these lesions.     

Cytologic and cytometric analysis of oral mucosa in Alzheimer's disease

OBJECTIVE: To analyze cytomorphologically the buccal mucosa of patients with Alzheimer's disease (AD). STUDY DESIGN: Brush biopsies were obtained from 10 patients with AD and 9 age-matched controls without neurologic symptoms from 3 distinct oral sites. RESULTS: A significant reduction in partially keratinized intermediate (red) cells was observed in the buccal mucosa of the AD group. In the AD group, parabasal cells from the floor of the mouth (p = 0.017) and buccal mucosa (p = 0.058) and red cells,from the tongue dorsum (p = 0.013) and buccal mucosa (p = 0.002), exhibited significantly greater nuclear areas. With regard to the nuclear to cytoplasmic (N:C) ratio, intermediate (red) cells from the buccal mucosa and tongue dorsum of AD individuals showed a decrease in this parameter (p <0.0001), while superficial (yellow) cells (from buccal mucosa) (p= 0.042) and parabasal (blue) cells (from the tongue dorsum) (p = 0.003) exhibited an increased N:C ratio. No significant differences were detected in the cells from the floor of the mouth. CONCLUSIONS: Our findings indicate that cytologic and cytometric changes were detectable in the exfoliative cytology of the buccal mucosa and tongue in the AD group.     

Candida biotypes in patients with oral leukoplakia and lichen planus

Prevalence of yeasts in 35 leukoplakia and 34 oral lichen planus patients was compared with that observed in persons without oral diseases. Serotype and morphotype were determined on Candida albicans isolates. Yeasts were isolated from the oral cavity specimens of 43.7% of the patients. C. albicans (serotype A) was the predominant species (76% in leukoplakia, 88.2% in lichen planus and 60.8% in healthy persons). Sixteen morphotypes were encountered on malt extract agar, being 732, 733, 734, 753 and 754 the most frequently found. Morphotypes SP1N and SP1Y were the most common on Sabouraud-trypheniltetrazolium agar (68.4% of the isolates from leukoplakia and 73.3% from lichen planus, but only 46.6% of the isolates from healthy oral mucosa showed SP1N morphotype). Presence of oral lesions was associated with a marked reduction in the yeast species and C. albicans biotypes, suggesting that C. albicans and particularly some of its biotypes, show a high potential of adaptation to the changes associated with the development of oral leukoplakia and lichen planus.     

成人口腔黏膜念珠菌病的多因素分析

为探讨影响成人口腔黏膜念珠菌病发生的因素,选取口腔黏膜念珠菌病、复发性阿弗他溃疡、扁平苔藓、舍格伦综合征、白斑、天疱疮、类天疱疮患者及口腔黏膜健康者为研究对象,详细记录731个样本的年龄、性别、吸烟、口腔卫生情况、抗生素使用、义齿、口腔黏膜疾病、全身疾病,对结果进行多元Logistic回归分析,得到影响口腔黏膜念珠菌病发生的因素有4类,分别为发病前使用抗生素、口腔卫生情况、口腔黏膜疾病、全身疾病,即多种因素的作用导致口腔黏膜念珠菌病的发生。     

Detection and identification of non-Candida albicans species in human oral lichen planus

Candida species were detected and identified in samples from the buccal mucosa, dorsal surface of the tongue and supragingival plaque of subjects with oral lichen planus (OLP). The Candida in the samples were cultured on selection agars, and identified by sequence analyses of 18S, 5.8S and 25/28S rRNA. The isolation frequency of Candida was higher in subjects with OLP than in those with healthy oral mucosa. Non-C. albicans were only isolated from people with OLP. These results support the notion that subjects with OLP are more likely to have oral colonization with Candida, and that non-C. albicans are specifically present in subjects with this condition.     

Trace metals in oral mucosa in relation to the lichen ruberplanus pathology. A preliminary study carried out by neutron activation analysis.

Lichen ruberplanus, contact allergy and hypersensitivity can be linked to oral exposure to metals released from metal alloys commonly used in dentistry. In this context neutron activation analysis was developed for the microdetermination of 36 elements in oral mucosa biopsies of two patients affected by lichen ruber planus and of five subjects as control group. In order to minimise metal contamination during sample collection, biopsies were taken by laser bistoury technique and then submitted to radiochemical neutron activation analysis (RNAA). Among the metals analysed, chromium showed obvious high concentration in gingival biopsies of the two pathological subjects compared to the corresponding tissues of control group. Cobalt and nickel were also determined in higher concentrations, but only in one of the oral mucosa of the two patients. The present findings way support the hypothesis concerning a potential link of lichen ruber planus condition with the exposure to Cr, Co and Ni as released into oral cavity from prosthodontic alloys.     

Salivary levels of tumor necrosis factor-alpha in oral lichen planus

OBJECTIVE: Oral lichen planus (OLP) is chronic inflammatory disease of the oral mucosa, presenting in various clinical forms. The etiology of OLP is still unknown but mounting evidence points to the immunologic basis of this disorder. AIM: Our study was undertaken to quantify the salivary levels of pro-inflammatory tumor necrosis factor-alpha (TNF-alpha) in the reticular and the erosive/atrophic forms of OLP, compared with age-matched healthy control volunteers. SUBJECTS AND METHODS: Whole saliva from 40 patients with active lesions of OLP, as well as from 20 healthy persons, was investigated for the presence of TNF-alpha by enzyme immunoassay. RESULTS: Salivary TNF-alpha levels were significantly increased in patients with OLP in comparison with healthy subjects. The presence of TNF-alpha showed positive correlation to clinical forms of OLP, being significantly higher in the erosive/atrophic type than in the reticular type of disease. CONCLUSION: Saliva provides an ideal medium for the detection of pro-inflammatory markers of the oral cavity. In patients with OLP, TNF-alpha levels in saliva are elevated, correlating with the severity of illness. Salivary TNF-alpha analysis may be a useful diagnostic tool and a potential prognostic marker in OLP.     

Malignancy-associated changes in breast tissue detected by image cytometry.

In several tissues, nuclear differences have been described in normal-appearing cells from patients with invasive carcinomas compared to cases without invasive carcinoma, a phenomenon known as malignancy-associated changes (MACs). The aim of this study was to determine the presence of malignancy-associated changes in breast tissue. Image cytometry was performed on Feulgen stained tissue sections of patients with usual ductal hyperplasia with (n = 30) or without (n = 41) adjacent invasive breast carcinoma. Nuclear features of normal-appearing cells as well as of usual ductal hyperplastic cells were separately compared between the two groups. Many features of normal-appearing epithelial cells were significantly different between cases with and without invasive cancer. Significant differences were also found by measuring ductal hyperplastic nuclei instead of normal-appearing nuclei. Cases with or without cancer could be distinguished with a classification accuracy of 80% by discriminant analysis using 2 nuclear features derived from ductal hyperplastic cells. In conclusion, image cytometry on breast tissue sections shows that malignancy-associated changes can be found in normal as well as in usual ductal hyperplastic breast cells. This could be clinically relevant for the detection of occult breast cancer, for the prediction of risk in these lesions, and to monitor the effect of chemopreventive agents.     

清热解毒祛风中药治疗口腔扁平苔癣的临床疗效及对患者外周血T淋巴细胞亚群的影响观察

目的：探讨养血解毒祛风中药治疗40例口腔扁平苔藓的临床疗效以及其对OLP患者外周血T淋巴细胞亚群的影响。方法：将80例扁平苔癣患者随机分为中药组40例和西药对照组40例。治疗组采用中药养血解毒祛风法辨证施治,对照组患者以西药治疗,用左旋咪唑50mg／次,3次／d,口服3天停4天一个月。结果：治疗组与对照组统计学上有显著性差异,中药治疗对患者T细胞亚群有很好的调节作用。结论：中药清热解毒祛风治疗口腔扁平苔癣有显著疗效。     

Frequency of micronucleated exfoliated cells in oral lichen planus

OBJECTIVE: The purpose of this study was to determine the frequency of micronucleated exfoliated cells (MEC) in atrophic and erosive oral lichen planus (OLP). MATERIALS AND METHODS: Twenty-two patients with atrophic and/or erosive OLP participated in this study. Lesions were scored ranging from 0 (no lesion) to 5 (large erosion) according to the severity and assessed for MEC. Exfoliated cells were obtained by swabbing the lesions and normal-appearing mucosa adjacent to the lesions. Swabbing was also performed in age-sex-matched normal individuals. Five hundred exfoliated cells were screened for nuclear anomalies including micronuclei, karyorhexis, pycnosis, and chromatid clumping. RESULTS: The severity score of OLP ranged from 2 to 4 with the average of 2. The frequency of MEC in OLP patients was 3.79% and 0.37% in the lesions and normal-appearing mucosa, respectively. In normal individuals, the frequency of MEC was also 0.37%. Using a paired t-test, it was found that the MEC frequency in the OLP lesions was significantly elevated (p<0.01) as compared to that in normal-appearing mucosa adjacent to lesions and that in normal individuals. There were no statistically significant differences in the MEC frequency of the three severity scores as analyzed by Kruskal-Wallis one way analysis of variance on ranks (p>0.05). CONCLUSION: This study revealed an increase in micronuclei in OLP lesions. The results indicate genotoxic damage in atrophic and erosive OLP.     

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.     

低功率氩离子激光治疗口腔癌前病变的临床研究

目的对氩离子激光治疗口腔癌前病变的治疗机理、照射功率、方法及疗效差异进行探讨。方法 对５３例口腔白斑和扁平苔藓患者应用Ａｒ＾＋激光治疗,检查比较患者治疗前后的临床变化。结果 口腔白斑３１例全部治愈,治愈率１００％;口腔扁平苔藓２２例治愈１３例,显效７例,无效２例,治疗有效率９０．９％,总有效率达９６．２％。结论 氩离子激光治疗口腔白斑和扁平苔藓等清表的口腔慢性病损,不仅疗效显著,操作简便,并发症少     

Altered expression of integrins and basement membrane proteins in malignant and pre-malignant lesions of oral mucosa

Integrins are transmembrane receptors that regulate cell-cell and cell-extracellular matrix (ECM) contact. In epithelial tissues, they interact with ECM components of the basement membrane (BM) to maintain the homeostasis and the architecture of the tissue. This interaction controls several cell functions such as adhesion, migration, proliferation, differentiation, and therefore has a key role in cancer development and metastasis. We studied the expression of integrins and ECM components of the BM by immunohistochemistry in frozen specimens of malignant squamous cell carcinoma (SCC), pre-malignant lesions of the oral mucosa (leucoplakia) and oral lichen planus. In invasive SCC, we observed altered polarity and distribution of alpha2beta1, alpha6beta4 and alpha3beta1 integrins, whereas in the in situ carcinoma alpha6beta4 and alpha3beta1 patterns only were altered. Immunostaining for ECM components such as Laminin-1 (Ln-1), Ln-5, and Collagen IV (Coll IV) was discontinuous and interrupted in invasive SCC, whereas it was normal in the in situ carcinoma. In both pre-malignant lesions and lichen planus specimens, integrins were expressed in a polarized manner in the presence of a normal BM, whereas were abnormally distributed in those tissues with altered staining patterns of the ECM components. In conclusion, we suggest that abnormal re-distribution of alpha3beta1 and alpha6beta4 integrins and expression of ECM components such as Ln-5 could play an important role in SCC invasion and metastasis.     

Buccal Spectral Markers for Lung Cancer Risk Stratification

Lung cancer remains the leading cause of cancer deaths in the US with >150,000 deaths per year. In order to more effectively reduce lung cancer mortality, more sophisticated screening paradigms are needed. Previously, our group demonstrated the use of low-coherence enhanced backscattering (LEBS) spectroscopy to detect and quantify the micro/nano-architectural correlates of colorectal and pancreatic field carcinogenesis. In the lung, the buccal (cheek) mucosa has been suggested as an excellent surrogate site in the “field of injury”. We, therefore, wanted to assess whether LEBS could similarly sense the presence of lung. To this end, we applied a fiber-optic LEBS probe to a dataset of 27 smokers without diagnosed lung cancer (controls) and 46 with lung cancer (cases), which was divided into a training and a blinded validation set (32 and 41 subjects, respectively). LEBS readings of the buccal mucosa were taken from the oral cavity applying gentle contact. The diagnostic LEBS marker was notably altered in patients harboring lung cancer compared to smoking controls. The prediction rule developed on training set data provided excellent diagnostics with 94% sensitivity, 80% specificity, and 95% accuracy. Applying the same threshold to the blinded validation set yielded 79% sensitivity and 83% specificity. These results were not confounded by patient demographics or impacted by cancer type or location. Moreover, the prediction rule was robust across all stages of cancer including stage I. We envision the use of LEBS as the first part of a two-step paradigm shift in lung cancer screening in which patients with high LEBS risk markers are funnelled into more invasive screening for confirmation.     

Karyometric marker features in tissue adjacent to in situ cervical carcinomas

Subtle changes in nuclear chromatin structure have been documented in the histologically normal mucosa adjacent to neoplastic lesions. To evaluate the expression of such "marker features" in tissue adjacent to squamous carcinomas in situ (CIS) of the uterine cervix, normal-appearing ectocervical tissues from five cases of CIS were compared with ectocervical tissues from control patients with squamous metaplasia. Formalin-fixed, paraffin-embedded tissue sections were Feulgen stained and analyzed with the microTICAS video microphotometer. Discriminant analysis revealed seven features that helped to distinguish nuclei from ectocervical tissue adjacent to CIS from those of control tissue. These features reflected changes in nuclear shape and chromatin distribution that were not detected by routine histopathologic analysis. The findings may reflect a subtle premalignant change in the apparently normal mucosa adjacent to a cervical neoplasm; they may also reflect the influence of the neoplasm on the adjacent mucosa.     

Analysis of oral microbial community and Th17‐associated cytokines in saliva of patients with oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory disorder of oral mucosa of unknown cause. Microbial infection and dysimmunity appear to play important roles in its pathogenesis. In this study, differences in genetic profiling of salivary microbial communities in two subtypes of OLP and healthy controls were evaluated by means of PCR‐denaturing gradient gel electrophoresis (DGGE). Additionally, ELISA was used to investigate the possible role of Th17 in lesion formation by detecting two related cytokines IL‐17 and IL‐23 in the saliva of OLP patients. When the DGGE profiles were analyzed, the bacterial populations were found to be significantly less rich in subjects with reticular and erosive OLP than in healthy controls. There was significantly less microbial diversity, as denoted by the Shannon index, in saliva samples from subjects with erosive OLP than in those from healthy controls. Cluster analysis and principal component analysis showed that the DGGE profiles formed distinctly group‐specific clusters. Salivary concentrations of IL‐17 in subjects with erosive OLP group were significantly higher than in those with reticular OLP and healthy controls. What's more, significantly positive correlations were observed between salivary IL‐17 concentrations and disease clinical scores. Microbial richness and diversity was negatively correlated with salivary IL‐17 concentrations. These results suggest there is significantly less salivary bacterial diversity and complexity in subjects with OLP han in healthy controls and that the shifted community composition is closely related to an immune cytokine, IL‐17.     

肥厚性扁平苔藓皮损中浸润淋巴细胞的研究

目的探讨淋巴细胞浸润在肥厚性扁平苔藓发病机制中的意义。方法采用免疫组织化学SP法对10例肥厚性扁平苔藓患者皮损、20例非肥厚性扁平苔藓患者皮损及10例正常人皮肤中CD3、CD4、CD8、CD20进行检测。结果所有患者皮损均以CD3^＋’T、CD4^＋T、CD8^＋T细胞浸润为主,散在CD20^＋B细胞浸润。肥厚性皮损中浸润T淋巴细胞数少于非肥厚性皮损（P〈0．01）,CD20^＋B细胞占浸润淋巴细胞百分率高于非肥厚性皮损（P〈0．05）。正常人皮肤仅在真皮偶见CD3^＋T细胞。结论肥厚性扁平苔藓皮损T淋巴细胞的减少,B淋巴细胞的增多表达可能与疾病的持续存在及局部过度增殖性改变密切相关。