Nuclear morphometry and estrogen receptors in infiltrating ductal carcinoma of the breast

In this study ten cases of breast infiltrating ductal carcinoma have been considered. In all of them the content of ER has been evaluated by using monoclonal antibodies. Five of them were ER positive and five were ER negative. For the morphometric study ten nuclei of each case have been considered. By using the S.A.M. (Shape Analytical Morphometry) work-station an analytical study of the nuclear shape was performed. The first step was the extraction of fundamental shape which describes the basic shape of original contour without its irregularities. It was obtained by using two parametric equations. The second step was the evaluation of shape asymmetry by S.A.E. (Shape Asymmetry Evaluator). Finally the contour irregularities were evaluated by Fourier analysis. Along with analytical parameters, dimensions (area, perimeter and maximum diameter) were considered too. All obtained data were submitted to univariate statistical analysis (Student's T test) to compare the two groups (ER positive and ER negative tumors). Area, perimeter and maximum diameter were significatively greater in ER negative cases while analytical parameters were not discriminant between the two groups.     

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.     

Analytical morphometry of sagittal fronto-facial profiles of 2nd-6th month human foetus

A prominent problem in the morphological study of developing biological structures is shape parametrization with the aim to evaluate transformations in a non subjective way. We carried out an analytical morphometrical study on a series of human embryos, by means of the S.A.M. (Shape Analytical Morphometry) software package. After standardization and normalization of the fronto-facial sagittal profiles, the analytical procedures applied were: 1) Fourier analysis: each profile was considered as an irregular but periodic function obtained by the sum of sinusoids of increasing order. 2) Shape Asymmetry Evaluation: couples of profiles are compared by means of "Janus" procedure; by a parabolic fitting we obtained parameters able to evaluate allometric and isometric differences between the two profiles. The preliminary results of the applied procedures indicated that rate and direction of allometric and isometric growth are not constant during the time.     

Proliferating activity in oral dysplastic leasions and squamous cell carcinomas

The aim of the study was the quantitative analysis of AgNORs in oral squamous cell carcinomas as well as in dysplastic epithelial changes accompanied and not accompanied by oral squamous cell carcinomas. AgNOR proteins were visualized in histological slides using silver impregnation technique according to D. Ploton. In each sample 100 cell nuclei were assessed. The study used 54 cases of proliferating oral epithelial changes divided into 3 groups: group I consisting of 13 cases of dysplastic lesions not accompanied by oral squamous cell carcinomas; group II (a total of 18 cases) containing dysplastic lesions situated in the vicinity of oral carcinomas and group III (23 cases) with oral squamous cell carcinomas. Statistically significant differences were found between groups with mild dysplasia and groups with severe dysplasia as well as squamous cell carcinomas. Statistical analysis did not show any differences in the number of AgNORs between squamous cell carcinomas and epithelial lesions with severe dysplasia. Our results demonstrate that the analysis of AgNORs expression can serve only as an additional parameter to evaluate the potential of malignant transformation.     

DNA content in human uterine cervical dysplasias.

The DNA content of nuclei extracted from separate biopsies of normal cervical epithelium and from dysplastic sites in the same patient was determined biochemically. Mean percentage increase in the DNA content of dysplasias (mild: 143.3 +/- 14.9; moderate: 225.8 +/- 51.1; and severe: 359.8 +/- 45.8) was found to be statistically significant (F-Test: p less than 0.001) over the normal values in control cervical tissues.     

Comparison of three different methods for automated classification of cervical cells.

Over 4600 exfoliated squamous cervical cells taken from appropriate Papanicolaou samples were classified as normal, mildly dysplastic, moderately dysplastic and severely dysplastic by an experienced cytopathologist. The slides were de-stained and subsequently re-stained with Feulgen Thionin-SO2 stain. Images of the nuclei were then captured, recorded and processed employing an image cytometry device. Automated classification of the cells was carried out using three different methods--discriminant function analysis, a decision tree classifier and a neutral network classifier. The discriminant function analysis method, which combined all dysplastic cells into an abnormal group, achieved a combined error rate of less than 0.4% for moderate and severe dysplastic cells, and less than 40% for mildly dysplastic cells. All three methods yielded comparable results, which approached those of human performance.     

Single-cell DNA measurement in hyperplastic, dysplastic and carcinomatous laryngeal epithelia, with special reference to the occurrence of hypertetraploid cell nuclei

Single-cell DNA fluorometry was performed on smear preparations from 57 histologically verified lesions from the vocal cords. Special attention was paid to the detection of hypertetraploid cell nuclei, which may be considered neoplastic markers, since they are not present in normal vocal cord epithelium. Hypertetraploid nuclei were not found in hyperplastic or mildly dysplastic epithelia but were found in 4 of 14 specimens of moderate dysplasia and in 5 of 19 specimens of severe dysplasia. The presence of hypertetraploid nuclei was associated with more frequent recurrences and progression of disease. Hypertetraploid cell nuclei were found in 9 of 15 invasive carcinomas and in this group were associated with a worse prognosis. The proliferative activity was 1.7% for hyperplasia and mild dysplasia, 3.1% and 2.8%, respectively, for moderate and severe dysplasia and 7.6% for squamous-cell carcinoma.     

Expression of cyclins A and E in melanocytic skin lesions and its correlation with some clinicopathologic features

Cyclins play a fundamental role in the cell cycle. Recent studies have focused on their role in the development of various malignancies. The objective of this study was to evaluate and compare the expression of cyclins A and E in common nevi, dysplastic nevi and malignant melanomas, and to investigate the relationship between cyclin expression and some pathological parameters such as tumor thickness, ulceration, regression, and mitotic rate, as well as several clinical and phenotypic parameters such as skin phototype, hair and eye color, number of nevi, personal or family melanoma history, and personal history of nonmelanoma skin cancer (NMSC). A total of 102 melanocytic skin lesions, including 30 common nevi, 38 dysplastic nevi and 34 melanomas, were examined. Expression of cyclins was detected by immunohistochemistry and quantified as a percentage of immunostained cell nuclei in each sample. Significant differences in expression of both cyclins were found between all lesion types: the median percentage of cyclin A-positive nuclei was 8.2% in melanomas, 3.4% in dysplastic nevi, and 0.95% in common nevi (p < 0.001). The corresponding percentages for cyclin E were 9.5%, 4.25% and 1.44% (p < 0.001). Expression of both cyclins was significantly higher among patients with a personal history of NMSC. Cyclin A was also significantly overexpressed in patients with a high total nevus count (TNC) compared to moderate and low TNC. Expression of cyclins did not significantly correlate with the other clinicopathologic features investigated. These findings indicate the possible involvement of cyclins A and E in the pathogenesis of malignant melanoma. Our results also show a potential diagnostic significance of these cyclins as markers allowing discrimination between dysplastic nevi and melanoma.     

Systolic time intervals in burn injury

Systolic time intervals were studied in 28 young, previously healthy burn patients (TBS 10-90%) on 145 occasions. A NEK 116 type 3 channel recorder of 100 mm/sec paper speed was used. Synchronous ECG recordings in lead II, PCG in the m "1" frequency band, and external carotid pulse tracings were recorded. RR, QS2, S1S2, LVET, PEP, PEP/LVET and QT, QTc, QS2-QT intervals were measured in 5-10 cardiac cycles in each of the examinations. Calculation of the additional parameters ICT, LVET/ICT, DT, EVR, PRP, EF and SV values were correlated to the extent of, and the time elapsed since, burn injury. The derivated parameters and statistical analysis were performed on a Commodore 64 type computer based on a software program. Elongation of QTc (greater than 440 msec) and frequent electrodynamic failure (QT-QS2 greater than 40 msec) especially during the first postinjury week were found characteristic in severe injury. In 53% the PEP/LVET ratio was less than 0.31, mean 0.32 +/- 0.093. In 21 cases of severe hypovolaemia the increase of PEP raised this value beyond 0.41. With the exception of 3 examinations, QS2I was normal or shortened. Calculated EF were normal or increased, SV reduced being interdependent with burn extent and shortening of DT. In patients with severe burns, oxygenization was inadequate (EVR less than 0.8); it showed an inverse correlation to HR. The simultaneously high LVET/ICT ratio and shortened ICT values pointed to an increase in contractility.     

Simultaneous quantification of nucleoproteins and comparison of methyl green-pyronin Y and Feulgen staining in sections of oral squamous cell carcinoma,dysplastic lesions and normal mucosa

A fundamental difference between normal cells and tumor cells is the proliferative activity of the nucleus and nucleolus, which increases progressively from normal to oral dysplastic mucosa to oral squamous cell carcinoma (OSCC). This activity is evaluated routinely using hematoxylin and eosin (H & E) staining, but in some cases, inter-observer variability occurs among pathologists. We evaluated cellular proliferation by staining sections with the methyl green-pyronin Y procedure and the Feulgen reaction. We also compared the efficacy of methyl green-pyronin Y and Feulgen staining for studying nuclear and nucleolar features in oral dysplastic mucosa and in different grades of OSCC. Sections cut from formalin fixed, paraffin embedded blocks of five normal mucosa, 15 dysplastic mucosa, 10 well-differentiated OSCC, 10 moderately differentiated OSCC and five poorly differentiated OSCC cases were stained with Hematoxylin and Eosin, methyl green-pyronin Y and the Feulgen reaction. The mean diameters of the nuclei and number of nucleoli showed significant differences. A progressive increase in diameter of the nucleus and number of nucleoli was observed from normal mucosa through poorly differentiated OSCC. We observed that methyl green-pyronin Y stain is more useful than Feulgen and hematoxylin and eosin for simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine examination.     

Nuclear S100A7 Is Associated with Poor Prognosis in Head and Neck Cancer

Background

Tissue proteomic analysis of head and neck squamous cell carcinoma (HNSCC) and normal oral mucosa using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and liquid chromatography-mass spectrometry, led to the identification of a panel of biomarkers including S100A7. In the multi-step process of head and neck tumorigenesis, the presence of dysplastic areas in the epithelium is proposed to be associated with a likely progression to cancer; however there are no established biomarkers to predict their potential of malignant transformation. This study aimed to determine the clinical significance of S100A7 overexpression in HNSCC.

Methodology

Immunohistochemical analysis of S100A7 expression in HNSCC (100 cases), oral lesions (166 cases) and 100 histologically normal tissues was carried out and correlated with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients. Overexpression of S100A7 protein was significant in oral lesions (squamous cell hyperplasia/dysplasia) and sustained in HNSCC in comparison with oral normal mucosa (p_trend<0.001). Significant increase in nuclear S100A7 was observed in HNSCC as compared to dysplastic lesions (p = 0.005) and associated with well differentiated squamous cell carcinoma (p = 0.031). Notably, nuclear accumulation of S100A7 also emerged as an independent predictor of reduced disease free survival (p = 0.006, Hazard ratio (HR = 7.6), 95% CI = 1.3−5.1) in multivariate analysis underscoring its relevance as a poor prognosticator of HNSCC patients.

Conclusions

Our study demonstrated nuclear accumulation of S100A7 may serve as predictor of poor prognosis in HNSCC patients. Further, increased nuclear accumulation of S100A7 in HNSCC as compared to dysplastic lesions warrants a large-scale longitudinal study of patients with dysplasia to evaluate its potential as a determinant of increased risk of transformation of oral premalignant lesions.     

Procedure for immunocytochemical detection of P16INK4A antigen in thin-layer, liquid-based specimens

OBJECTIVE: To develop a procedure for the immunocytochemical detection of P16INK4A in ThinPrep specimens. STUDY DESIGN: Archived ThinPrep, liquid-based cervical/endocervical cytology specimens (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) diagnosed as LSIL, HSIL and WNL were resampled and fixed in 95% ethanol for at least three days. Rehydration and endogenous peroxidase blocking of both ThinPreps and formalin-fixed, paraffin-embedded tissues were accomplished on a Leica Autostainer (Leica, Deerfield, Illinois, U.S.A.). Microwave antigen retrieval with CitraPlus (Biogenex, San Ramon, California, U.S.A.) was performed using a Panasonic microwave oven (Matsushita Cooking Appliances, Franklin Park, Illinois, U.S.A.) on the high setting twice for five minutes each. After cooling for 20 minutes and undergoing a buffer rinse, the slides were placed in a Dako autostainer (Dako-USA, Carpinteria, California, U.S.A.). The P16INK4A primary antibody, clone E6H4 (MTM Laboratories, Heidelberg, Germany) was diluted 1:200 in antibody diluent buffer. Detection was accomplished with a mouse non-avidin-biotin EnVision+ polymer (Dako). The expression of P16INK4A in ThinPreps and corresponding biopsies were scored by two pathologists. A ThinPrep case was scored as positive if it contained > 10 abnormal cells with nuclear and cytoplasmic immunocytochemical staining. Corresponding biopsies were scored as exhibiting negative, sporadic, focal or diffuse staining, as described by Klaes et al, Overexpression of P16INK4A as specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri (Int J Cancer 2001;92:276-284). RESULTS: The P16INK4A antibody assay was positive in 14 of 19 (73.68%) LSIL ThinPrep cases and in 25 of 26 (96.15%) HSIL ThinPrep cases. Thirty-eight of the 39 (97.44%) biopsies corresponding to the positively stained ThinPreps also were positive, with a staining score of at least focal positivity in the dysplastic regions. The P16INK4A antibody assay was negative in 5 of 19 (26.32%) LSIL ThinPrep cases and negative in 1 of 26 (3.85%) HSIL ThinPrep cases. The six biopsies corresponding to the negative ThinPreps were similarly negative. The two cytologic specimens diagnosed as WNL were negative for P16INK4A, as were two tissue control cases with benign diagnoses. Nondysplastic squamous epithelium, identified in 17 biopsy cases, did not stain, nor did nondysplastic squamous cells identified in ThinPrep cases. Sporadic staining of bacteria, inflammatory cells and occasional endocervical glandular cells was identified. CONCLUSION: P16INK4A expression in ThinPrep specimens correlates with tissue expression of P16INK4A, as implemented in the above protocol. P16INK4A may thus serve as a surrogate marker in gynecologic cytology for high-risk HPV infection and for the development of cervical neoplasia.     

Flow cytometric DNA analyses of epithelial dysplasia of the esophagus

OBJECTIVE: To investigate, with flow cytometry, DNA aneuploidy as a marker of early carcinogenesis in dysplastic esophageal lesions. STUDY DESIGN: DNA content of exfoliated cells from 789 cases of esophageal dysplasia (including mild dysplasia, 195 cases; moderate dysplasia, 383 cases; and severe dysplasia, 211 cases) was determined with a FACS 420 flow cytometer. RESULTS: Cellular DNA content was closely related to the severity of dysplasia. The carcinogenesis rate in patients with dysplasia showed that DNA aneuploidy was significantly higher than in patients showing DNA diploidy. CONCLUSION: DNA aneuploidy in dysplastic lesions is a very important early signal of carcinogenesis. Patients with dysplastic lesions showing DNA aneuploidy should be treated and closely followed.     

Computer simulation modelling and visualization of 3D architecture of biological tissues

Recent technical improvements, such as 3D microscopy imaging, have shown the necessity of studying 3D biological tissue architecture during carcinogenesis. In the present paper a computer simulation model is developed allowing the visualization of the microscopic biological tissue architecture during the development of metaplastic and dysplastic lesions.The static part of the model allows the simulation of the normal, metaplastic and dysplastic architecture of an external epithelium. This model is associated to a knowledge base which contains only data on the nasal epithelium. The latter has been well studied by numerous authors and its lesional states are well known. An inference engine allows the initialization of the static model parameters. A statistical comparison between simulated epithelia and real epithelia is achieved by adjusting the parameter values during the simulation.The dynamic part of the model allows the simulation of a growth process on a 3D representation based on the static model. The main hypothesis is that nasal epithelium is submitted to a continuous transformation from normal to cancer through metaplasia and dysplasia. The evolution of each cell (represented by its nucleus) depends on its local environment and also on its heritage from its mother-cell.Simulation of tissue renewal of the nasal pseudostratified epithelium has been achieved. The evolution from normal to hyperplasia has been simulated. After modification of the cell cycle modelling, the simulation of the development of metaplastic foci has been obtained.     

Karyometric image analysis for intraepithelial and invasive cervical lesions

OBJECTIVE: To derive an objective, numeric measure for the progression of intraepithelial and invasive squamous cell cervical lesions. STUDY DESIGN: Thin-layer cervical cytology preparations from colposcopically confirmed normal cervix, low grade squamous intraepithelial lesions, high grade squamous intraepithelial lesions and carcinoma were identified from a cross-sectional study. Fifty-nine cases representing 4 diagnostic categories were selected, and 2,375 nuclei from epithelial cells representative of the diagnostic category were randomly selected for imaging and measurement from these cases. Additionally, 1,378 visually normal appearing intermediate cells from low and high grade squamous intraepithelial lesions, as well as from carcinoma cases, were identified for analysis. The nuclei were quantitatively characterized, and discriminant analyses were performed to derive a progression curve from normal cytology to carcinoma. RESULTS: The lesion signatures show a clear increase in nuclear abnormality with increasing progression. A progression curve was derived based on mean discriminant function scores for each diagnostic category and on the mean nuclear abnormality values for the nuclei in each category, as expressed by their deviation in feature values from normal reference nuclei. CONCLUSION: A numeric assessment of lesion progression for cervical precancerous and cancerous lesions based on karyometric measurements is possible and may provide an objective, precise characterization of each lesion as well as a basis for improved performance in automated cytology-based cervical cancer screening.     

Progression curves for endometrial lesions

OBJECTIVE: To derive a numeric measure for the progression of endometrial lesions as a baseline study for an eventual assessment of chemopreventive intervention efficacy. STUDY DESIGN: Tissue sections from normal endometrium at the proliferative and secretory phase, simple hyperplasia, atypical hyperplasia from cases free of concomitant adenocarcinoma and adenocarcinoma of the endometrium were recorded at high spatial resolution. Six cases from each diagnostic category were chosen as "typical," and 60 epithelial nuclei were randomly selected for measurement for each case. Discriminant analyses were carried out to derive a direction of progressive change in feature space and to correct the progression curve for the presence of cells not expressing progressive change among the random sample of nuclei. RESULTS: A well-conditioned progression curve was derived based on the mean discriminant function scores for each diagnostic category and the mean nuclear abnormality of the nuclei in each category, as expressed by their deviation in feature values from normal reference nuclei. The lesion signatures showed a clear trend toward extension into the range of higher nuclear abnormalities with increasing progression. There was an indication that abnormal endometrial lesions may comprise cases with distinctly different degrees of nuclear abnormality. CONCLUSION: A numeric assessment of lesion progression for endometrial lesions, based on karyometric measurements, is possible. The data suggest that additional analysis may provide further characterizing information for individual lesions.     

Towards 3-D modelling of epithelia by computer simulation.

The present work is a preliminary step towards dynamic 3-D modelling by computer graphics simulation of the structure of normal and pathological epithelia, using an expert system. In its present state, Esexsy (Epithelium Simulation by EXpert SYstem) allows the construction, through iterative steps, of a simple 3-D representation of the nasal epithelium, based on the positions, sizes and shapes of nuclei. The iterative process is based on statistical comparisons between distributions of parameter values calculated from real (2-D) histological sections and those issued from an equivalent computer 'section' through the simulated 3-D image. We show the results of attempts at simulating normal, metaplastic and dysplastic states of the nasal epithelium, the latter two being characterized by a progressive architectural disorganization, accompanied by nuclear size/shape alterations. The representation takes into account the size, shape, orientation and spatial arrangement of nuclei, with one or several layers from the basal lamina to the lumen. A modified Poisson point process is used at present to position the nuclei, which are modelled by bi-axial spheroids (from prolate to oblate through spherical), with random orientation and size/shape deviations. It should be possible to use the same computer program to simulate other types of epithelia and to achieve increasingly realistic representations by incorporating, notably, nuclear deformations and chromatin texture.     

Morphometric discrimination between syndromic (Gorlin) and nonsyndromic keratocysts.

OBJECTIVE: To search for morphologic nuclear features in the epithelial lining of odontogenic keratocysts to differentiate simple from Gorlin syndrome cysts. STUDY DESIGN: Five cases of syndrome-associated keratocysts and five of simple ones were studied. Thirty nuclei from the epithelial basal layer for each case were analyzed by the shape analytical morphometry (SAM) software system to quantitatively evaluate nuclear dimensions (area, perimeter, diameter), contour irregularities and nuclear shape asymmetries. Results were subjected to Student's t test and cluster analysis. RESULTS: Values of nuclear dimensions were very close in both groups of keratocysts, without any significant statistical differences. The variables related to nuclear profile irregularities, as well as those describing nuclear asymmetry, showed significantly higher values (P < .001) in syndromic cysts. Cluster analysis produced two different clusters by using variables related to nuclear contour irregularities. CONCLUSION: Preliminary results indicate the existence of nuclear morphologic differences between simple and syndromic cysts.