Cytological analysis of surface epithelial layers of oral mucosa

Various methods of cytological analysis of surface epithelial layers of the buccal mucosa are compared in patients in need of dentures. Different informative value of cytological prints, scrapings, and washings off the oral cavity for analysis of exfoliated epitheliocytes is shown. The described methods help to evaluate the regeneration potentialities of the buccal mucosa epitheliaum in the course of treatment. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 1, pp. 113–116, January, 2000     

Cytology,flow cytometry,image analysis,and interphase cytogenetics by fluorescence in situ hybridization in the diagnosis of transitional cell carcinoma in bladder washes: A comparative study

The diagnosis of transitional cell carcinoma (TCC) in bladder washes is a diagnostic challenge to cytology. This study assessed the role of flow cytometry (FCM), image analysis (IA), and interphase cytogenetics by fluorescence in situ hybridization (FISH) as adjuncts in the cytodiagnosis of TCC in bladder washes. Forty separate samples of bladder washes were prospectively evaluated by conventional cytology (CY), FCM, IA, and FISH, and the results were compared with the subsequent surgical biopsy specimens which revealed 26 TCC (3 GR I, 6 GR II, 17 GR III) and 14 benign lesions. Using histology as the “gold standard” and following the previously published criteria for detection of TCC by CY, FCM, IA, and FISH, the concordance rates between histology and CY, FCM, IA, and FISH were 75, 74, 89, and 83%, respectively. CY, FCM, IA, FISH, and histology were concordant in 54% of the cases. The sensitivity of CY, FCM, IA, and FISH were 61, 72, 91, and 73%. respectively, while the specificity were 100, 80, 83, and 100%, respectively. The combined sensitivity of all the parameters was 96%. Interestingly, the false positive cases by FCM and IA showed cystitis. We conclude that IA has the highest sensitivity in detecting TCC in bladder washes followed by FISH, FCM, and CY, while CY and FISH have the highest specificity. This study indicates that FCM, IA, and FISH are useful adjuncts to cytology in the diagnosis of TCC in bladder washes. The finding of DNA-aneuploidy in cystitis warrants further investigation. © 1995 Wiley-Liss, Inc.     

Solid and papillary epithelial tumour of the pancreas: Cytological case study with ultrastructural and flow cytometric evaluation

An intraoperative cytological diagnosis of a rare solid and papillary epithelial tumor of the pancreas was made at laparotomy in a 15-year-old Indian female. The characteristic cytological features were the presence of numerous large branching papillary clusters with central vascular stalks lined by multiple layers of uniform bland cells. Perivascular metachromatic material was prominent on Romanovsky stain. Ultrastructural identification of endocrine and exocrine features supports a multipotential cell origin in small pancreatic ductules. DNA analysis, not previously reported for this tumour, demonstrated a diploid population of tumour cells with a low S-phase fraction (0.4%). This would explain the bland nuclear morphology and favourable prognosis with a high rate of surgical cure for this neoplasm.     

A simplified method for the routine culture of normal oral epithelial (NOE) cells from upper aerodigestive tract mucosa

A method has been established for the routine culture for normal oral epithelial (NOE) cells based on simple methodology and commercially available materials. Normal mucosa was obtained from surgical resections of cancer patients, oropharyngeal mucosa, or pediatric tonsillectomies, anterior tonsillar pillar. NOE cells were allowed to outgrow using explant outgrowth techniques on whole mucosa or the dispased epithelial component. Outgrowth in AmnioMax-C100 medium (Gibco) followed by cell passaging in KGM (Clonetics) using Primaria (Falcon) tissue culture dishes produced reproducible growth, a reflection of a high rate of cell proliferation. Using this technique, cells maintain log phase growth for 2 cell passages, a minimum of 10–20 population doublings, and over 10⁸ cells can be easily obtained from most specimens, if desired. The growth potential of NOE cells from individual clinical specimens can be predicted by cell size on a particle counter. If the mean population diameter is approximately 15 m or less, cells exhibit good log phase growth whereas if the diameter is greater than 16 um, growth is poor. NOE cells cultured by this method show typical epithelial morphology, have desmosomal junctions, rest on an extracellular matrix, are positive for cytokeratin expression and are growth inhibited by TGF-, a normal inhibitory growth regulator. In summary, using this technique, epithelial cells from human oral mucosa can be easily generated in sufficient quantity and quality for cell biological, biochemical and molecular studies.Abbreviations NOE normal oral epithelia - CE cornified envelopes - IHC immunohistochemistry - TGF- transforming growth factor beta     

Application of flow cytometry for biomarker-based cervical cancer cells detection

The Pap test used for cervical cancer screening is subjective, labor-intensive, and has relatively low sensitivity and specificity for the detection of underlying clinically significant lesions. The objective of this study is to develop a biomarker/flow cytometry-based approach for cervical cancer screening. Immunofluorescence technology to quantify cervical cell expression of two biomarkers p16(INK4A) and Mcm5 was developed and evaluated by both microscopy and flow cytometry. The capability of using biomarker/flow cytometry approach to detect rare-event dysplastic cells in a large background of benign epithelial and inflammatory cells was evaluated. The results indicate that flow cytometry could detect 0.01% dysplastic cells in a background of normal cervical epithelial cells with the combination of the two biomarkers. Thirty-two clinical specimens were used to test the biomarker/flow cytometry-based approach and the results were compared with the liquid-based cervical cytology. The experiment yielded 100% sensitivity and 93% specificity with reference to the liquid-based cervical cytology. This study indicates the promise of using multi-color fluorescence flow cytometry for biomarker-based cervical cancer screening. This molecular-based, potentially high-throughput and automated method is expected to provide an alternative/auxiliary means of cervical cancer screening.     

Evaluation of tissue engineered models of the oral mucosa to investigate oral candidiasis

Candida albicans is a commensal organism that can be isolated from the majority of healthy individuals. However, in certain susceptible individuals C. albicans can become pathogenic leading to the mucocutaneous infection; oral candidiasis. Murine models and in vitro monolayer cultures have generated some data on the likely virulence and host factors that contribute to oral candidiasis but these models have limitations. Recently, tissue engineered oral mucosal models have been developed to mimic the normal oral mucosa but little information is available on their true representation. In this study, we assessed the histological features of three different tissue engineered oral mucosal models compared to the normal oral mucosa and analysed both cell damage and cytokine release following infection with C. albicans. Models comprised of normal oral keratinocytes and a fibroblast-containing matrix displayed more similar immunohistological and proliferation characteristics to normal mucosa, compared to models composed of an oral carcinoma cell line. Although all models were invaded and damaged by C. albicans in a similar manner, the cytokine response was much more pronounced in models containing normal keratinocytes. These data suggest that models based on normal keratinocytes atop a fibroblast-containing connective tissue will significantly aid in dissecting the molecular pathogenesis of oral candidiasis.     

A dual-species microbial model for studying the dynamics between oral streptococci and periodontal pathogens during biofilm development on titanium surfaces by flow cytometry

The association of the pioneer organisms Streptococcus mutans ATCC 25175 or Streptococcus oralis ATCC 9811 with secondary colonizers Fusobacterium nucleatum ATCC 25586 or Porphyromonas gingivalis ATCC 33277 during biofilm development on titanium surfaces was evaluated by flow cytometry (FCM) using specific polyclonal antibodies.ELISA and FCM were employed, revealing high antibody sensitivity and specificity. Biofilm formation of four dual-species combinations was analyzed by crystal violet staining, while the association between streptococci and periodontal pathogens was assessed using FCM. Dual-species association between S. oralis and P. gingivalis or F. nucleatum showed a proportional decrease in S. oralis during biofilm development, with a concomitant increase in P. gingivalis or F. nucleatum. This trend was not observed in either of the dual-species associations of S. mutans with the periodontal pathogens.Our dual-species microbial model, which employed FCM, proved to be useful in the study of partnerships between bacteria in oral associations, showing that the presence of primary colonizers is required for the establishment of secondary colonizers in biofilms. The proposed experimental approach is technically simple to prepare and analyze, and also proved to be reproducible; hence, it is well-suited for investigating the development and dynamics of oral communities.     

Perspectives of African Americans and dentists concerning dentist-patient communication on oral cancer screening

OBJECTIVE: Mortality rates for oral cancer have not improved appreciably in decades, with Blacks less likely than others to survive 5-years post-treatment. Oral cancer is the fifth most common cancer among African American males, representing a pressing public health concern. This study compared how dentists and African American adults view the current state of dentist-patient communication regarding oral cancer and its detection. METHODS: Five focus groups with 56 African American adults and two focus groups with 17 dentists were conducted in order to compare responses regarding oral cancer information needs and dentist-patient communication on oral cancer screening. RESULTS: African American adults showed little knowledge about oral cancer and cancer screening, and great need for information. However, dentists reported rarely engaging in information exchange with their patients even while performing the examination. CONCLUSION: African Americans' request for screening information and dentists' reticence about performing the screening and initiating communication with patients indicate a need for both public education on oral cancer and improved continuing education courses for dentists. PRACTICE IMPLICATIONS: The dental care community should develop clear guidelines for communicating with patients about oral cancer and engage in continuing education on oral cancer screening procedure. Increased public health efforts aimed at increasing oral cancer awareness and knowledge among the public are warranted.     

Computer-assisted analysis of cell proliferation markers in oral lesions

Abnormalities in any component of the cell cycle regulatory machine may result in oral cancer, and markers of cell proliferation have been used to determine the prognosis of tumor progression. The aim of this study was to determine whether silver-stained nucleolar organizer region (AgNOR) and Ki-67 measurements could improve the assessment of growth rates in oral lesions. Eighty-three oral biopsies were studied, 20 of which were classified as fibrous inflammatory hyperplasia (FIH), 40 as leukoplakia (LKP) and 23 as oral squamous cell carcinoma (OSCC). Within the LKP group, 22 out of 29 biopsies were diagnosed as non-dysplastic leukoplakia (LK) and 18 as dysplastic leukoplakia (DLK), presenting discrete, moderate and severe dysplasia. Ki-67 immunolabeling of the lesions increased steadily in the following order: FIH, DLK, LK and OSCC, indicating that Ki-67 is a good marker for predicting the proliferative fraction among benign, premalignant and malignant oral lesions. The median values of AgNOR parameters indicate that the morphometric index gives better results regarding the proliferative rate than the numerical one. A series of linear regressions between AgNOR parameters and Ki-67 showed positive associations. We conclude that a combination of Ki-67 and morphometric AgNOR analyses could be used as an aid in the determination of the proliferative status of oral epithelial cells in oral cancer.     

Interepithelial cells of the oral mucosa in mice

Summary Non-epithelial mesenchymal and neuroectodermal cells occur between the keratinocytes in the stratified squamous epithelium of the oral mucosa. These cells cannot be classified adequately by light microscopy. In the present study the oral mucosa of the lip, cheek and tongue of 50 mice were studied by light and electron microscopy. 3,025 mononuclear interepithelial cells were documented and analysed.Monocytogenic macrophages, plasma cells and mast cells were not found interepithelially and cannot be regarded as a regular constituent of the epithelium. Only a few neuroectodermal cells — in mice these are exclusively Merkel cells, with no melanocytes — were localized in the epithelium. The majority of the interepithelial cell population is made up of lymphocytes (22.8%) and Langerhans cells (56.8%). They are an integral constituent of the epithelium. Lymphocytes with rounded and indented nuclei can be identified. The larger and dendritic Langerhans cells are a specific cell of squamous epithelium and also occur in the oral mucosa. Not all cells which feature the cytological characteristics of Langerhans cells contain Langerhans or Birbeck granules. Accordingly these granules cannot be considered an exclusive identification characteristic. Two types of Langerhans cells can be differentiated. 80.9% have the more or less typical appearance known from the epidermis and were termed macrophagocytoid Langerhans cells. The nuclei are irregularly indented and moderately heterochromatic. 19.1% possessed conspicuous large, spherical, euchromatic nuclei and an electron-lucent cytoplasm. These were termed reticuloid Langerhans cells. About 20% of the interepithelial cell population could not be identified, neither as typical lymphocytes nor as Langerhans cells. These were small to medium sized cells with deeply indented cerebriform strongly heterochromatic nuclei. They are similar to the Sézary cells or mycosis fungoides cells of epidermotropic human T-cell lymphomas. The lymphocytic nature of these cells has been confirmed. It seems likely that differentiation of lymphocytes to cerebriform cells occurs within the epithelium. It is further discussed whether cerebriform cells are precursors of Langerhans cells, a conclusion suggested morphologically by transitional forms. This would imply that Langerhans cells originate from lymphocytes, and that the cerebriform cell is an intermediate step of differentiation. The microenvironment of the squamous epithelium may play a role in the process of differentiation, which could explain the epitheliotropy of lymphocytes. The possibility is considered that Langerhans cells and interdigitating reticulum cells of the T-cell area of lymph nodes are identical. The close functional cooperation of Langerhans cells, lymphocytes, and interdigitating reticulum cells in immunological defenses against external antigens is discussed.The authors wish to express their sincere appreciation to Miss P. Starck and Miss I. Brandt for invaluable technical assistance in this project.     

Comparative molecular and histological grading of epithelial dysplasia of the oral cavity and the oropharynx

Histological grading of epithelial dysplasia in the oral cavity and oropharynx is used to predict the risk for cancer and to determine the treatment strategy. This grading, however, is subjective and not well reproducible. Recent publications have shown that molecular markers are promising in cancer risk assessment. The aim of the present study was to compare classical histological and molecular grading and to relate these to the proliferation rate by quantitative assessment of Ki-67 staining. Forty-three samples were analysed from the margins of patients who had undergone resection of their squamous cell carcinoma in the oral cavity/oropharynx. Three experienced pathologists performed the histological grading. With the consensus score, 12 samples were classified as normal and 31 as dysplastic (21 mild, six moderate, and four severe). Loss of heterozygosity (LOH) was assessed in the same samples with 15 microsatellite markers at chromosomes 3p, 9p, 17p, 8p, 13q, and 18q, and was present in 28 of the 43 samples. Twenty-four of the 28 cases (86%) with LOH were classified as dysplastic and four as normal. All ten samples with moderate and severe dysplasia and 14 of 21 samples with mild dysplasia contained LOH. In four of 12 biopsies classified as normal, LOH was found. A very striking and significant difference of the Ki-67 index was observed between LOH-positive and LOH-negative cases, 36.6 +/- 11.1% versus 19.4 +/- 2.8% positive cells, respectively. In mild dysplasia, 13 of 14 lesions containing LOH had a higher Ki-67 index than all seven lesions without LOH. Thus, in the oral cavity/oropharynx, LOH is more frequently found in the histologically higher-grade lesions (moderate dysplasia or worse) and in the lower grade lesions when a high proliferation rate is present. Assessment of proliferation with Ki-67 is a better surrogate for LOH than histological grading.     

Value of multi-parameter flow cytometry immunophenotyping in T/NK-cell neoplasms in cytology specimens: A retrospective study in Chinese patients

BackgroundInnate limitations of morphological diagnosis of T/NK-cell neoplasms mean that they can be misdiagnosed or missed, especially when mixed with a variety of benign and reactive conditions. The aim of this study was to investigate the application value of multiparameter flow cytometry immunophenotyping (MFCI) in screening and diagnosing T/NK-cell neoplasms with cytology specimens.Material and methodsThe clinical and pathological characteristics of 1028 newly diagnosed cases from Fudan University Shanghai Cancer Center who provided a cytology specimen between June 2010 and January 2016 with correlated histology diagnosis and clinical confirmation were retrospectively reviewed. MFCI was used for screening, diagnosis and typing. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in diagnosis of T/NK-cell neoplasms were calculated.ResultsThere were 606 males and 422 females in 1028cases, with a mean age of 47.5 years (range 9–86 years). Specimens used for cytologic diagnosis included 996 FNAs, 2 US-FNAs, 13 EUS-FNAs and 17 effusions. Screening for types of lymphoma of MFCI, 139 (13.52 %) cases were T/NK cell lymphoma, 3 (0.29 %) cases were B cell lymphoma T-NHL and B-NHL coexist. A total of 146 suspected T/NK-cell neoplasms were screened out (sensitivity = 94.64 %, specificity = 95.63 % PPV = 72.60 %, NPV = 99.32 %) by MFCI, with 112 (76.71 %) histologically confirmed cases and 6 (4.11 %) false-negative cases identified (3 cases diagnosed as B-cell neoplasms and 1 case as T-cell neoplasm with B-cell neoplasm, which also were confirmed by gene rearrangement. 2 cases were suspicious T-cell–immunophenotypic abnormalities). When used at the diagnostic level, a total of 88 T/NK-cell neoplasms were identified (sensitivity = 68.75 %, specificity = 98.80 %, PPV = 87.50 %, NPV = 96.28 %) with 11 false-positive cases recognized, 9 of which showed typical immunophenotypic T-cell neoplasms features, and 2 exhibited aberrant T immunophenotype.ConclusionsMFCI has high sensitivity and specificity in the screening and diagnosis of T/NK-cell neoplasms and may be useful as an alternative diagnosis method in cytology specimens.     

Oral epithelial atypical changes in apparently healthy oral mucosa exposed to smoking,alcohol, peppers and hot meals,using the AgNOR and Papanicolaou staining techniques

To evaluate cytological atypical changes in apparently healthy oral mucosa exposed to smoking, alcohol, hot meals, and peppers using the AgNOR and Papanicolaou methods. A total of 180 individuals were evaluated, of which 60 were smokers, 34 were alcohol users, 52 were habitual peppers and hot meal (exposed) consumers, 24 were non‐exposed, and 10 were patients with Oral Squamous Cell Carcinoma (OSCC), as an internal control. Cytological materials were obtained by brushing of buccal mucosa, on the border of the tongue and on the floor of the mouth, and participants underwent the Papanicolaou test for cytological changes and AgNOR staining for evaluation of the mean number of AgNOR dots per nucleus. SPSS program was used to perform the Pearson chi‐square test. The 95% confidence level, Odds Ratio (OR), and the 95% Confidence Intervals (CI) were used. The features of cytological atypia were verified among 10 individuals, including 5 smokers, 2 alcohol users, 2 hot meals and peppers consumers, and one non‐exposed. For atypia among tobacco smokers, the adjusted Odds Ratio (OR) and the 95% CI were found to be 2 (0.246–16.24). Increased keratinization was detected among 27 (45%) of the smokers (P < 0.0001), 17 (32.7%) of the pepper and hot meals consumers (P < 0.005), 4 (11.8%) of the alcohol consumers, and among 2 (3.7%) of the non‐exposed group. Statistical analyses revealed a greater mean number of AgNORs per nucleus in smokers (3.68) followed by (2.82) alcohol consumers, compared to the habitual peppers and hot meal consumers (2.28) and the non‐exposed group (2.00). What's more, 80% of the smears with cytological atypia were identified with 6 ± 2 AgNOR mean count. The increase of the variables suggests that the evaluation of epithelial atypical changes in individuals exposed to smoking and alcohol carcinogens may be a useful screening tool. While hot meals and peppers didnot seem to be a risk for oral mucosal proliferation, they increased the potency of keratinization and infection. Diagn. Cytopathol. 2010. © 2009 Wiley‐Liss, Inc.     

Evaluation of p16/Ki-67 dual-stain cytology performed on self-collected vaginal and clinician-collected cervical specimens for the detection of cervical pre-cancer

ObjectivesTo compare the performance of dual immunostaining of p16INK4a and Ki-67 proteins performed on self-collected vaginal specimens and clinician-collected cervical specimens, and to evaluate the performance of this technique in predicting high-grade disease.MethodsWomen aged 30–59 years (n = 1005) were recruited at two well-women clinics in Papua New Guinea. Each woman provided both cervical and vaginal specimens that were tested for high-risk human papillomavirus (hrHPV) DNA using the Xpert HPV Test (Cepheid) at point of care. A subset of paired cervical and vaginal specimens (n = 243) were selected to undergo CINTec® PLUS (Roche) p16/Ki-67 dual-stain cytology and liquid-based cytology (LBC).ResultsFifty-five pairs (22%) were excluded from further analysis because the smears were not assessable. Of the 189 remaining paired specimens, 74 pairs (39.1%) were positive for one or more hrHPV genotypes. When comparing results of the dual stain, the overall percent agreement, positive and negative percent agreements and κ value between the cervical and vaginal specimens were 87.8% (CI 82.3–92.1%), 64.6% (CI 49.5–77.8%), 95.7% (CI 91.0–98.0%) and 0.65 (CI 0.51–0.79%) respectively. The sensitivity of the dual stain performed on the cervical specimen to predict high-grade disease, determined by LBC, was superior to that of the dual stain performed on the vaginal specimen: 100% (CI 84.6–100%) versus 68.2% (CI 45.1–86.1%).ConclusionAlthough further evaluation may be warranted, these findings indicate that dual-stain testing of vaginal specimens cannot be advocated as part of cervical screening programmes in low- and middle-income countries. However, dual-stain cytology performed on cervical specimens may have a role in quality assurance in such settings.     

肺癌患者口腔脱落上皮细胞微核的观察

作者测定了肺癌患者口腔脱落上皮细胞微核率、出现微核人员率、多微核上皮细胞出现率，并对化疗前后肺癌患者各项指标进行了比较，结果表明肺癌组各项指标均显著高于对照组（Ｐ＜０．０１），而化疗后肺癌患者各项指标又显著高于肺癌组（Ｐ＜０．０１），提示微核检测可用于癌肿及抗癌化疗药物的染色体畸变观察。     

Generation of homologous cell pairs using the oral lymphatic system

The purpose of this study is to establish in vivo and in vitro models for studying lymphatic metastasis of squamous cell carcinoma (SCC). Three cell lines CAL-27, Tca-83, and HeLa were injected into the tongue of nude mice. Forty days after injection, we could isolate cells of 2 homologous cell lines LN-CAL-27 and LN-HeLa from lymph node metastasis lesions. Then, the homologous cell pairs were compared by the CCK-8 assay, wound healing assay, real-time PCR, western blot, and animal experiments. The results showed that all the three cell lines could be used to establish lymphatic metastasis animal models, and the lymphatic metastasis process was observed clearly. In addition, the homologous cell pairs performed differently from parent lines with respect to biological behavior and lymphatic metastasis-related gene and protein expression. In conclusion, CAL-27, Tca-83, and HeLa cells could be used to simulate the lymphatic metastasis process of oral cancer in vivo. Furthermore, the homologous cell pairs (CAL-27 and LN-CAL-27; HeLa and LN-HeLa) are potential tools for in vitro investigation of the mechanisms underlying metastasis.     

Developing the Quantitative Histopathology Image Ontology (QHIO): A case study using the hot spot detection problem

Interoperability across data sets is a key challenge for quantitative histopathological imaging. There is a need for an ontology that can support effective merging of pathological image data with associated clinical and demographic data. To foster organized, cross-disciplinary, information-driven collaborations in the pathological imaging field, we propose to develop an ontology to represent imaging data and methods used in pathological imaging and analysis, and call it Quantitative Histopathological Imaging Ontology – QHIO. We apply QHIO to breast cancer hot-spot detection with the goal of enhancing reliability of detection by promoting the sharing of data between image analysts.