The classification and grading of the noninvasive, intraepithelial neoplasms of the urothelium are based on the morphological pattern of growth, i.e., papillary and flat (and endophytic), and on their degree of architectural and cytologic abnormalities. Recent advances in the morphological, molecular and quantitative evaluation of these lesions have contributed to the refinement of the current classification and grading schemes. However, some controversies on the precise criteria and terminology, especially when the papillary lesions are concerned, are still present.
OBJECTIVE:To develop expert systems for classification of follicular thyroid tumor at a preoperative stage.STUDY DESIGN:Fine needle aspiration biopsy of the thyroid gland with a histologic conclusion of follicular cancer and follicular adenoma were the object of the morphometric study. General sample size was 4500 nuclei and 3000 aggregates.RESULTS:Quantitative regularities of pathologic changes in thyrocyte nuclei and aggregates in follicular cancer and follicular adenoma were revealed. Threshold values and weighting coefficients of quantitative features of thyrocyte nuclei and aggregates characterizing cancer made the basis of the two expert systems. Expert systems included standard 2-D S-matrix containing threshold values of nuclei and aggregates in cancer and their weighting coefficients as well as 1-D scientific X-matrix designed for filling with quantitative features of the studied object. The diagnosis was verified by the value of a diagnostic index by means of comparing feature values in the corresponding elements of S- and X-matrices. After that, a diagnostic index was calculated taking into account the features' weighting coefficient.CONCLUSION:The developed expert systems based on a set of quantitative features of thyrocyte nuclei and aggregates will allow assessing the malignant potential of a follicular thyroid tumor at a preoperative stage.
OBJECTIVE:To establish the karyometric characteristics of the two main nuclear phenotypes in cutaneous squamous cell cancer (cSCC) lesions.STUDY DESIGN:The clinical materials comprised 75 cases of cSCC, 38 with aggressive lesions and 37 with nonaggressive lesions. High-resolution images of 100 nuclei per case were recorded. Data were partitioned into four subgroups covering the range of lesion progression. Four discriminant functions were derived to distinguish aggressive from nonaggressive lesions. The most typical nuclei from the phenotype predominant in aggressive lesions and nonaggressive lesions were separated out by thresholding on the discriminant function score axes. For these homogeneous sets of nuclei the karyometric features were computed.RESULTS:The nuclear populations in cSCC lesions are a very heterogeneous set. There are two axes of dispersion, along the line of lesion progression and between aggressive and nonaggressive lesions. The analysis faces the difficulty that lesions from both diagnostic categories contain nuclei of the same two phenotypes with the difference between categories consisting only of differences in proportion of the two phenotypes.CONCLUSION:The nuclei of the aggressive phenotype I and nonaggressive phenotype II have substantially different chromatin patterns and can be distinguished with > 90% correct recognition rate.
OBJECTIVE:To assess the potential contribution of nuclear morphometry to the differential diagnosis of renal epithelial tumors with eosinophilic cytoplasm, including chromophobe renal cell carcinoma (ChRCC), the eosinophilic variant of clear cell renal cell carcinoma (EoRCC), and oncocytoma.STUDY DESIGN:A total of 24 tumor tissue samples diagnosed as ChRCC, the EoRCC, or oncocytoma constituted our series. Eight geometric features such as nuclear area, nuclear perimeter, and circular form factor were measured and compared among the groups.RESULT:On the basis of nuclear morphometry, measurements of eight geometric features significantly differ among these problematic eosinophilic renal epithelial neoplasms (p < 0.005).CONCLUSION:Because of their different biologic behaviors, the exact discrimination of the renal epithelial tumors with eosinophilic cytoplasm is crucial. However, this distinction can sometimes be problematic even for highly experienced pathologists. Our results suggest that the morphometric nuclear shape descriptors may be used as an ancillary method in their differential diagnosis.
OBJECTIVE To evaluate the role of micronuclei (MN) scoring in urinary cytology of bladder transitional cell carcinoma (TCC). STUDY DESIGN Eighty cases of freshly voided urine samples were used for scoring of MN using acridine orange (AO)-stained smears under fluorescence microscope and routine Papanicolaou-stained smears under light microscopy. MN score for cytologic lesions was compared. RESULTS In AO-stained slides, the mean MN scores were 0.171 +/- 0.395 and 1.489 +/- 1.871 in smears with "no malignant cells seen" and TCC cases, respectively. In Papanicolaou-stained slides the mean MN scores were 0.194 +/- 0.419 and 1.84 +/- 1.876 in smears with "no malignant cells seen," and TCC cases, respectively. Independent sample t test showed significant difference of MN scoring in Papanicolaou-stain and AO stain in no malignancy vs. TCC. Pearson's correlation test showed strong positive correlation between mean MN score by AO and Papanicolaou stains and Pearson's correlation coefficient. CONCLUSION MN score was significantly high in TCC cases, which may be due to chromosomal damage in bladder carcinoma. In difficult cases, MN may help distinguish benign from malignant cells in urine samples. Routine Papanicolaou stain may also be used for MN score because DNA-specific AO-stained smear gives almost similar results.
OBJECTIVE: To test a Cell Profiler pipeline for automated counting and characterization of gamma-H2AX foci in color images of human cultured cells.STUDY DESIGN: A431 cells were irradiated and stained for gamma-H2AX foci detection. Sets of color images were analyzed visually, and findings were compared with those using FociCounter and CellProfiler software.RESULTS: The CellProfiler pipeline includes some proprieties not present in FociCounter, such as the automatic detection of nuclei, the detection of touching nuclei and the rejection of nuclei that touch the border of the image. The time required for manual operation is associated with the number of images analyzed visually or by FociCounter but not for the CellProfiler program. CellProfiler reduced manual operation time and is about 4 times faster than semiautomatic detection using Foci-Counter and 10 times faster than visual counting.CONCLUSION: We conclude that CellProfiler and FociCounter are reliable tools for measuring gamma-H2AX foci. However, CellProfiler overcomes the limitations of the FociCounter program and is able to detect nuclei automatically, saving considerable manual operation. (Anal Quant Cytol Histol 2012;34:66-71)
There exists a wide and heterogeneous spectrum of spindle cell lesions that can originate from the prostatic stroma. This includes benign and malignant neoplasms as well as tumorlike conditions. Their diagnosis in small biopsy specimens is often difficult because of the potential for sampling error. The utility of ancillary studies, including immunohistochemistry, is often limited and the main criteria for diagnosis are the morphologic findings by routine hematoxylin and eosin stain.
OBJECTIVE:To evaluate DNA damage and cellular death in exfoliated buccal mucosa cells from healthy individuals following dental X-ray exposure, to compare effects of different types of radiographic techniques using the buccal cytome assay and to standardize the staining procedure.STUDY DESIGN:This study comprised 90 patients, categorized into 3 groups subjected to intraoral periapical radiography, conventional orthopantomogram and digital orthopantomogram X-ray exposure. Exfoliated oral mucosa cells were collected immediately before the X-ray and 10 days later and stained using DNA-specific stains and nonspecific DNA stains.RESULTS:The results indicated no statistically significant differences in micronucleated cells before and after dental X-ray exposure. On the other hand, X-ray exposure did increase other nuclear alterations closely related to cytotoxicity, such as karyorrhexis, pyknosis and karyolysis.CONCLUSION:These data indicate that dental radiography may not induce chromosomal damage, but it is able to promote cytotoxicity.
OBJECTIVE Deregulated cell proliferation is a hallmark of cancer, and Ki67 immunostaining can be used to identify proliferating cells. Evaluation of cell proliferation may have utility as a biomarker of epithelial malignant transformation risk. To date, most analyses of Ki67 staining have been restricted to semiquantitative estimations of the degree of staining or the measurement of the fraction of Ki67-positive cells within the epithelium. We sought to develop a robust, objective means of quantitatively evaluating Ki67 immunostaining for lung precancerous lesions. STUDY DESIGN We quantified the spatial distribution of Ki67-expressing cells within the epithelium by means of (1) a cell-based Voronoi tessellation and (2) a basement membrane-referenced distance transform. This was undertaken in a large cohort of 613 lung biopsy sections representing normal, hyperplasia, squamous metaplasia and mild, moderate and severe dysplasia. For each section 21 features quantifying different aspects of the Ki67 staining were calculated. Intraobserver and inter-observer variation were recorded for a subset of the biopsy sections. We examined the behavior of each feature with respect to histopathological grade. RESULTS These measures demonstrated that proliferation is generally limited to layers 2, 3 and 4 of the epithelium (layer 1 being the basal layer). The proliferation in the basal layer is limited and does not increase with increasing grade of dysplasia. Interobserver and intraobserver effects on these features were assessed, and several were more robust with respect to measuring Ki67 expression pattern than the commonly used fraction of Ki67-positive cells. CONCLUSION Many of these quantitative features showed associations with histological grade that were as strong as the association that exists based on the fraction of Ki67-positive cells while being much more robust to interobserver- and intraobserver-associated variations. The measured spatial distribution of proliferating cells statistically demonstrated asymmetric cell division behavior in cells in the basal layer, a pattern attributed to stem cells giving rise to transient amplifying cells.
OBJECTIVE:Color deconvolution relies on determination of unitary optical density vectors (OD(3D)) derived from pure constituent stains initially defined as intensity vectors in RGB space. OD(3D) can be defined in polar coordinates (phi, theta, radius); always being equal to one, radius can be ignored. Easier handling of unitary optical density 2D vectors (OD(2D)) is shown.STUDY DESIGN:OD(2D) pure stains used in anatomical pathology were assessed as centroid values (phi, theta) with a measure of variance: inertia based on arc lengths between centroid value and sampled points. These variables were plotted on a stereographic projection plane. In order to assess pure stains OD(2D), different methods of sampling RGB pixels were tested and compared: (2) direct sampling of nuclei from preparations using (a) composite H&E and (b) hematoxylin only and (2) for any pure stain RGB image, different associated 8-bit masks (saturation, brightness and RGB average) were used for sampling and compared. Behaviors of phi, theta and inertia were obtained by moving threshold in 8-bit mask histograms. Phi and theta stability were tested against variable light intensity during image acquisition and by using 2 different image acquisition systems.RESULTS:The more saturated RGB pixels are, the more stable phi, theta and inertia values are obtained. Different commercial hematoxylins have distinct OD(2D) characteristics. UltraView DAB stain shows high inertia and is angularly closer to usual counterstains than ultraView Red stain, which also has a lower inertia. Superior accuracy is expected from the latter stain. Phi and theta OD(2D) values are sensitive to light intensity variation, to the used imaging system and to the used objectives. An ImageJ plugin was designed to plot and interactively modify OD(2D) values with instant update of color deconvolution allowing heuristic segmentation.CONCLUSION:Utilization of polar OD(2D) eases statistical characterization of OD(3D) vectors: conditions of optimal sampling were demonstrated and various factors influencing OD(2D) stability were explored. Stereographic projection plane allows intuitive visualization of OD(3D) vectors as well as heuristic vectorial modification. All findings are not restricted to anatomical pathology but can be applied to bright field microscopy and subtractive color applications in general.
OBJECTIVETo introduce a new field of multifractal spectrum in distinguishing between endometrial well-differentiated adenocarcinoma (WDAC) and complex atypical hyperplasia (CAH).STUDY DESIGNThirteen cases of CAH and 16 of WDAC were selected from radical hysterectomy specimens, and multifractal spectrum was measured from at least 4-5 representative digitized images of each case. The data were collected from f(alpha) vs. alpha curves. The values of alpha max, alpha min, and their difference delta alpha (alpha max-alpha min) were recorded and the data compared.RESULTSThe mean +/- SD of alpha max, alpha min, and delta alpha of CAH were 2.36357 +/- 0.111623, 1.71357 +/- 0.032160, and 0.64214 +/- 0.094248, respectively. The mean +/- SD of alpha max, alpha min, and delta of WDAC were 2.50640 +/- 0.104545, 1.72100 +/- 0.036436, and 0.77620 +/- 0.108268, respectively. The mean of alpha max, alpha min, and delta alpha of WDAC were higher than in CAH. Mann-Whitney U test showed significant difference (p < 0.0001) of alpha max and delta alpja of WDAC and CAH.CONCLUSIONMultifractal dimension is significantly different in WDAC and CAH. The multifractal dimension is a new area in pathology. This study demonstrates the potential usefulness of multifractal analysis in histopathology.
OBJECTIVE:To analyze whether cell proliferation and apoptotic indexes in needle biopsies with prostate cancer Gleason 6 or 7 can identify more objectively Gleason score 6 or 7 in needle biopsy samples.STUDY DESIGN:Seventy patients diagnosed by needle biopsy and treated by radical prostatectomy were included. Fifty cases were Gleason score 6 and 20 were Gleason 7. Twenty-two cases were organ-confined and 48 nonorgan-confined. Histologic sections from needle biopsies were stained for cell proliferation using the MIB 1 (Ki67) antibody and in situ end-labeling technique for apoptosis and recorded as the percentage of positive cells. Statistical analysis included Student's t-test, Pearson's test, and logistic regression analysis.RESULTS:We found increased apoptotic and proliferation indexes from Gleason 6 to 7, and from organ-confined to non-organ-confined. Apoptotic index was not associated to stage or Gleason score. We identified an association between cell proliferation and Gleason score indicating that higher proliferation index is associated with a higher probability of presenting a Gleason score 7. There was no association between pathologic stage and cell proliferation.CONCLUSION:Cell proliferation and apoptosis can distinguish a subset of aggressive lesions in needle biopsies with Gleason 6 or 7 prostate adenocarcinoma.
OBJECTIVETo explore the possible role of osteopontin (OPN) in chronic plaque psoriasis and understand the role of inflammation in psoriasis.STUDY DESIGNWe investigated 36 patients with chronic plaque psoriasis using immunohistochemical technique for evaluation of OPN immunolocalization compared to normal skin biopsies of 10 normal subjects representing the control group.RESULTSOPN was expressed in the epidermis of all specimens, both in the psoriasis group and the control group without any significant differences except for the tendency of psoriatic lesions to show more cytoplasmic and nuclear pattern of OPN staining (55.56%) compared to normal skin (20%). Epidermal strong and diffuse immunostaining of OPN was associated with the severity of psoriasis, and there was a correlation of the intensity of OPN expression with the density of the dermal inflammatory infiltrate.CONCLUSIONOur study suggests that OPN is involved in the pathophysiology of psoriasis, and a possible association with disease severity. OPN acts by different mechanisms through its expression by lesional keratinocytes, inflammatory cells, and endothelial cells. Nuclear localization of OPN may have a more significant role in the pathogenesis of psoriasis.
OBJECTIVE To evaluate prognostic impact of maspin expression in patients with resected non-small cell lung cancer (NSCLC). STUDY DESIGN From 1996 to 2001, 439 patients underwent radical surgery for NSCLC at the Polytechnic University of the Marche Region. Maspin expression was detected as cytoplasmic and nuclear staining of neoplastic cells. For cytoplasmic staining, cases were classified as negative, low positive, and high positive. In positive cases, intensity of staining was also considered and scored. A similar classification was used for nuclear staining, but intensity was not considered. RESULTS The analysis showed that smoking history, pathologic stage of disease, N status, histologic grading, sex, and Eastern Cooperative Oncology Group performance status had a prognostic impact on overall survival (OS). Expression of maspin was also found to be an independent prognostic factor. A statistically significant longer OS was seen in patients with higher compared with lower expression of nuclear maspin, and poorer OS was present in patients with a higher intensity of cytoplasmic staining. Nuclear expression of maspin was also found to be an independent prognostic factor at multivariate analysis. CONCLUSION Results suggest that overexpression of maspin correlates with favorable prognosis in NSCLC. and may be a useful clinical marker.
OBJECTIVE: To determine an optimal prostate cancer gene 3 (PCA3) cutoff in predicting prostate cancer in Italian patients undergoing first or repeat biopsy.STUDY DESIGN: In this observational multicenter study 1,246 men with elevated prostate specific antigen (PSA) and negative digital rectal examination, with prostate biopsy after PCA3 assessment, were divided into two groups submitted to PCA3 testing before or after previous negative biopsies. Ideal PCA3 cutoff was identified using area under the curve of the receiver operating characteristic analysis. Various cutoff values were used to determine the best predictive score. Univariate and multivariate logistic regression models compared age, PSA, free-PSA, and PCA3 score to predict prostate cancer.RESULTS: PCA3 cutoff 39-50 had the highest accuracy in the repeat biopsy group in which cutoff of 39 could have avoided 51.9% negative repeat biopsies, eventually missing 7.8% of cancers (all low risk); cutoff of 50 would have prevented 56.5% of negative repeat biopsies, missing 29 tumors (10.3%), 5 potentially aggressive. The PCA3 test performed poorly in the first biopsy group.CONCLUSION: We confirm the usefulness of PCA3 in Italian men with a previous negative biopsy. We achieved the best performance at a cutoff of 39. PCA3 did not perform better than PSA in non biopsy-selected men. (Anal Quant Cytol Histol 2012;34:96-104)
OBJECTIVETo study the performance values of a set of five immunohistochemical markers involved in cell cycle regulation as a potential aid in the differential diagnosis between squamous intraepithelial lesions (SILs) and normal or benign conditions of the uterine cervix.STUDY DESIGNResults from immunohistochemical evaluation of p16, cyclin D1, p53, Ki67, and ProEx C markers and human papillomavirus genotyping were collected from a previous study performed on 37 normal or benign cervices, 39 low grade SILs and 73 high grade SILs. A multivariate analysis was used to examine the specific diagnostic value of each marker and to ascertain those most relevant for SIL diagnosis. For markers with good data fit, sensitivity, specificity, accuracy, area under the receiver operating characteristic curve, and integrated discrimination improvement, were calculated.RESULTSAmong individual markers, ProEx C showed the best specificity; p16 displayed the highest sensitivity and area under receiver operating characteristic curve for SIL diagnosis. Integrated discrimination improvement demonstrated that the p16 plus ProEx C model has better discrimination capacity than p16 plus Ki67 or ProEx C plus Ki67.CONCLUSIONUse of ProEx C alone or in combination with p16 could provide useful information for SIL diagnosis.
OBJECTIVE To compare the frequency of micronuclei and metanucleated anomalies in the oral mucosa of smokers, alcoholic smokers, and nonsmokers. STUDY DESIGN Three groups were studied: group I, 15 smokers; group II, 16 alcoholic smokers; and group III, 20 nonsmokers. Three smears from the lateral left border of the tongue were processed for Feulgen staining. A minimum of 300 cells per participant were examined for the quantification of micronuclei and metanucleated anomalies. RESULTS The Kruskal-Wallis test showed no significant difference in the frequency of micronuclei (p = 0.602) or karyorrhexis (p = 0.114) among the three groups, but there was a significant difference in the frequency of broken eggs, binucleated cells, and karyolysis (p = 0.001). Spearman's correlation indicated an influence of the number of cigarettes per day on micronuclei frequency. Tobacco caused significant alterations in the exfoliative cytology (broken eggs, binucleated cells, and karyolysis) of chronic smokers, but not in the frequency of micronuclei or karyorrhexis, despite the observation of a larger absolute number of micronuclei in group II. CONCLUSION The action of genotoxic agents (tobacco and alcohol) causes alterations in the frequency of micronuclei and metanucleated anomalies.
OBJECTIVE:To evaluate peritoneal fluid hemosiderin-laden macrophages (H-LMs) in patients with endometriosis compared to controls.STUDY DESIGN:Consecutive series of 46 patients during a year undergoing laparoscopy for benign gynecologic conditions were included. The presence of H-LMs in peritoneal fluid was evaluated. We compared clinical factors in patients with or without endometriosis in respect to H-LM status. To assess the potential of H-LMs to diagnose endometriosis, the sensitivity and specificity were calculated.RESULTS:Patients with endometriosis were significantly more likely to have positive H-LM test results than controls (p = 0.0013). The presence of H-LM has a low sensitivity of 52% but an acceptable specificity of 87% in diagnosing endometriosis. The presence of H-LMs was not related with any other of the clinical factors studied.CONCLUSION:The presence of specific findings of H-LMs related to endometriosis strongly suggests abnormalities in peritoneal iron metabolism.
For several decades, metastatic renal cell carcinoma (mRCC) was considered resistant to therapy and thus linked to poor prognosis. Recent years have seen a complete revolution of the therapeutic landscape, with the introduction of new target therapies that have radically changed treatment strategies in this setting. These new agents have proved to be effective if directed mainly against the angiogenic pathway. The vascular endothelial growth factor family is, in fact, the main target of a number of new drugs, such as monoclonal antibodies and different generations of tyrosine-kinase inhibitors that are currently approved or in different stages of clinical trial for the treatment of mRCC patients.
