Stereological estimates of nuclear volume correlated with histopathological grading and prognosis of bladder tumour

The aim of this retrospective study is to provide morphometric data which make grading of urinary bladder tumours objective and reproducible by stereological estimation of nuclear volume using the principle of estimating of the volume of particles of arbitrary shape. The study includes 92 specimens: 12 from normal bladder mucosa, and 80 from bladder tumours (15 grade I, 45 grade II, 19 grade III and one grade IV according to Bergkvist et al. 1965). After standard fixation, embedding, sectioning and hematoxylin-eosin staining, an unbiased estimate of the mean volume of nuclei sampled with a change proportional to the volume: (Formula: see text) was calculated. Here l0 is the length of the intercept through a test point hitting a nucleus measured in a random direction through the test point. The weighted means of nuclear volume in bladder tumours are spread over a wide range and show a strong correlation with the Bergkvist grade. Moreover, the relationship between the weighted mean volume of nuclei in bladder tumours and the prognosis is very good. Only one of 35 patients with a mean nuclear volume below 300 micron 3 died of bladder cancer whereas 18 of 19 patients with a mean nuclear volume above 500 micron 3 developed invasives tumours or died of their disease. This simple and fast estimate of nuclear volume seems to provide objective data of high prognostic value.     

Heterogeneic expression of blood group A and H isoantigens in bladder tumors: association with nuclear volume

Intratumor heterogeneity is a major problem in immunodiagnosis and treatment of carcinomas. To elucidate the well-known heterogeneity in transitional-cell carcinomas of the ability to express blood group ABO isoantigens, a stereological estimate of the mean nuclear volume in areas expressing blood group antigens was compared to the estimate from areas of identical pathological grade at which antigen expression was deleted. Four microscopic fields were examined from antigen-positive and four from antigen-negative areas in sections from 21 blood group O and 20 blood group A individuals. The sections were stained before examination by an indirect peroxidase method using monoclonal anti-H and anti-A antibodies. The mean nuclear volume increased, as expected, with increasing pathological grade. In blood group O individuals the mean nuclear volume was 241.5 microns 3 in antigen-positive areas and 338.2 microns 3 in antigen-negative areas (2p less than 0.0005) of identical pathological grade. In group A individuals the mean nuclear volume was 217.1 microns 3 in positive areas and 351.1 microns 3 in corresponding negative areas (2p less than 0.0025). The variation in volume parameter was essentially caused by a true variation between tumors (greater than 82%). The results indicate a complex biological mechanism associated with the cellular ability to express blood group antigens.     

Nuclear volume in type I gastric intestinal metaplasia

OBJECTIVE: To retrospectively compare nuclear size of epithelial cells in type I intestinal metaplasia (IM) from various pathologic lesions of gastric mucosa. STUDY DESIGN: Endoscopic mucosal biopsies with type I IM from intestinal type gastric carcinoma (n = 25), chronic gastritis (n = 40) and benign ulcer (n = 32) cases were analyzed. After standard fixation, embedding, sectioning, routine hematoxylin and eosin and alcian blue--periodic acid--Schiff reaction (pH 1.0 and 2.5) staining were used for identification of IM. The mean point sampled nuclear intercept was estimated by the original test system and 100x objective at a total magnification of 1,200x. To obtain the mean nuclear volume, the cubed nuclear intercept was multiplied by pi/3. In each case 100 epithelial cell nuclei were analyzed. RESULTS: In type I IM in gastric carcinoma cases there was significantly greater nuclear volume (118.34 +/- 10.32 micron 3) than in type I IM in other pathologic states of gastric mucosa (77.72 +/- 8.58 micron 3). CONCLUSION: The karyometric findings of the present study suggest a difference between type I IM found in benign pathologic states and type I IM found in gastric mucosa surrounding carcinoma, despite morphologic and histochemical similarities. Nuclear volume may be used in early detection of precancerous states of gastric mucosa.     

Mean nuclear volume in ovarian papillary serous cystadenocarcinoma

OBJECTIVE: To measure mean nuclear volume (MNV), estimated by the stereologic intercept method, in ovarian serous carcinomas and to compare that between omentum-positive cases (cases with omental metastasis) and -negative cases. STUDY DESIGN: This retrospective study consisted of 29 cases (stage I and II = 13 cases, and stage IIIb = 16 cases) of ovarian papillary serous cystadenocarcinoma. MNV of all cases (at primary and metastatic sites) was estimated with an image cytometer. The nuclear point intersection method was used to measure nuclear volume. Mean intercepted diameters of at least 50 nuclei were measured. MNV was correlated with histologic grade, International Federation of Gynecology and Obstetrics (FIGO) staging of ovarian malignancy and primary tumor versus metastatic deposits. RESULTS: MNVs estimated in omentum-negative (FIGO stage I and II) and omentum-positive (FIGO stage IIIb) cases were 1,022.48 +/- 608.45 and 2,152 +/- 1,317.51 microns 3 (P = .05, Student t test), respectively. Significant differences in MNV were also observed among the different grades of tumor. However, no significant difference was observed between MNVs of primary and metastatic tumors in omentum-positive cases. CONCLUSION: Estimates of MNV on conventional histopathology sections may provide objective and useful criteria for relatively subjective histopathology grading and staging (FIGO stage I and II vs. IIIb).     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

Stereologic estimation of mean nuclear volume in well-differentiated adenocarcinoma and carcinoma in situ of the gallbladder

OBJECTIVE: To investigate mean nuclear volume of cells in well-differentiated adenocarcinomas (20 cases) and carcinoma in situ (20 cases) of the gallbladder by the principle of estimation of the volume of particles with arbitrary shapes. STUDY DESIGN: Hematoxylin and eosin-stained, 4-micron-thick, vertical sections from formalin-fixed, paraffin-embedded tissue blocks were analyzed by using a projection microscope with a 100:1 oil immersion objective (NA 1.3); the final magnification was 2,500:1. The measurements were carried out in 10 microscopic fields for each slide. Mean nuclear volume was obtained by the stereologic method of point-sampled intercepts for vertical sections. RESULTS: Mean nuclear volume in well-differentiated adenocarcinomas (127.67 +/- 46.95 micron 3) was significantly larger than in carcinoma in situ (69.17 +/- 15.74 micron 3) (P < .000001). CONCLUSION: Stereologic estimation of mean nuclear volume may be helpful in the discrimination of malignant and borderline lesions of the gallbladder.     

Value of quantitative cytologic studies in the grading of papillary tumors of human urinary bladder

Mitotic indices and nuclear volumes were determined in 10 papillomas, 20 grade I, 20 grade II and 20 grade III papillary carcinomas of the urinary bladder. The mean mitotic indices were 1.22% in papillomas, 2.77% in grade I carcinomas, 8.2% in grade II carcinomas and 15.25% in grade III carcinomas. The nuclear volumes showed a gradual shift to larger values paralleling the histological degree. It is suggested that these objective parameters might be especially useful for the cytological assessment of exfoliated tumor cells.     

Prognostic significance of mean nuclear volume in pancreatic adenocarcinoma

OBJECTIVE: To evaluate the correlation of stereologically estimated mean nuclear volume of tumor cells with other clinicopathologic prognostic features and survival in pancreatic ductal adenocarcinoma. STUDY DESIGN: The study included 27 patients with primary pancreatic ductal adenocarcinoma. A stereologic method proposed by Gundersen et al was used for the estimation of mean nuclear volume in hematoxylin and eosin-stained histologic sections of each case. Mean nuclear volume values were compared statistically with histopathologic prognostic feature groups and survival. RESULTS: The mean nuclear volume values of tumor cells ranged from 296.83 to 982.79 microns 3 (mean, 633.906 +/- 212.310). Higher values of mean nuclear volume were significantly related to advanced tumor stage and the presence of distant metastasis (Kruskal-Wallis, P = .036; Mann-Whitney U, P = .020). In contrast, nodal stage, tumor grade, perineural invasion, lymphatic and blood vessel invasion, and size of tumor showed no statistical relation to mean nuclear volume of tumor cells. Mean nuclear volume was found to be a significant predictor of survival in univariate analysis (P = .016). CONCLUSION: Estimation of mean nuclear volume may help in predicting the extent of disease and clinical behavior in pancreatic adenocarcinoma.     

Mean nuclear volume in squamous cell carcinoma of the vulva

OBJECTIVE: To compare mean nuclear volume (MNV) estimated by the stereologic intercept method in lymph node-positive and -negative cases of squamous cell carcinoma of the vulva. STUDY DESIGN: This retrospective study consisted of 53 cases (lymph node metastasis, n = 19; cases without lymph node metastasis, n = 34) of squamous cell carcinoma of the vulva. MNV was estimated with the help of an image cytometer. The nuclear point intersection method was used to measure MNV. The mean nuclear volumes of both lymph node-positive and -negative cases were compared. RESULT: MNV in the lymph node-negative and -positive cases was 717.0 +/- 533.1 and 1,961.4 +/- 1,369.6 microns 3, respectively (P < .000, Mann-Whitney U test). There was a significant difference in MNV between the 2 groups of tumors. CONCLUSION: The observations from the present study suggest that estimation of MNV of malignant squamous cells from the vulva on conventional histopathology sections may provide an objective and useful diagnostic tool in predicting lymph node metastasis.     

Changes in nurse cell nuclei during synchronous infection with Trichinella spiralis

The rate of enlargement of nuclei was determined on 4-microns-thick sections of synchronously infected mouse thigh muscle. Normal muscle nuclei had a geometric mean volume of 84 microns and a range of 42-170 microns 3. At days 5, 6, 7, 8, and 10 and 6 mo after infection, mean nuclear volume was 177 (100-315) microns 3, 254 (140-462) microns 3, 278 (172-447) microns 3, 681 (407-1,138) microns 3, 512 (326-804) microns 3, and 509 (298-870) microns 3, respectively. Size of nuclei for any given day followed a log normal distribution. On days 7 and 8 after infection, 31% of enlarged nuclei had 2 nucleoli, whereas only 15% had 2 nucleoli on day 10. One percent of enlarged nuclei in 6-mo-old nurse cells had double nucleoli. The number of enlarged nuclei in 6-mo-old nurse cells was determined from serial sections of infected tongue muscle. Each nurse cell contained an average of 40 enlarged nuclei. Sixty-four percent of nurse cells examined (n = 55) had between 30 and 60 enlarged nuclei. However, there was great variation in the range (7-142). These results are discussed in relation to the development of the nurse cell.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Cytodifferentiation of polar plant cells: Formation and turnover of endoplasmic reticulum in root statocytes

By application of cytochalasin D (10 micrograms/ml) the distribution and turnover of endoplasmic reticulum (ER) in root statocytes of cress (Lepidium sativum L.) was studied. After 7 min of incubation, the distal ER complex, in 20-h-old control statocytes consisting of stacked cisternae, was disintegrated and redistributed. The amyloplasts sedimented into the most distal part of the cell. When the incubation time was increased up to 4 h, ER was formed near the nucleus and accumulated at the proximal cell pole. Thus microfilaments are suggested to be involved in (i) stabilization of the distal ER complex and (ii) the ER translocation from the forming site into the distal cell pole. By morphometric measurements the volume of story II (story III) statocytes was calculated to be 2400 microns3 (3000 microns3), containing an ER area of 1824 microns2 (2400 microns2). From the net ER formation rate of 5.2 microns2/min (story II) and 4.6 microns2/min (story III) and the net decrease rate of 23 microns2/min (story II) and 39.2 microns2/min (story III), a total ER formation rate of about 27 microns2/min (story II) and 43 microns2/min (story III) was estimated.     

Micropapillary variant of transitional cell carcinoma of the urinary bladder: a report of three cases with cytologic diagnosis in urine specimens

BACKGROUND: Micropapillary transitional cell carcinoma is a recently described, aggressive variant of bladder cancer. Its cytologic features in urine have not been previously characterized. CASES: Three cases illustrate the urinary cytologic features of this high grade urothelial carcinoma and its concurrent biopsy findings. This tumor is similar to low. grade urothelial lesions of the bladder, tends to present as micropapillary clusters in urine and yet has high grade nuclear features within these clusters that help with the differential diagnosis of a flat, high grade urothelial carcinoma. CONCLUSION: The micropapillary type of transitional cell carcinoma is a distinct morphologic entity with an aggressive clinical course. Recognizing its presence in urinary cytology, albeit a rare occurrence, is important in distinguishing this lesion from the more indolent, low grade papillary lesions and high grade urothelial carcinomas, which continuously shed single malignant urothelial cells.     

Proliferating cell nuclear antigen in normal urothelium and urothelial lesions of the urinary bladder: a quantitative assessment using a true color image analysis system

To evaluate proliferating cell nuclear antigen (PCNA) staining for assessing proliferative activity in routine pathology specimens of urinary bladder, the bladder carcinoma cell line J82 and a total of 122 specimens of normal bladder and urothelial lesions were stained with the antibody clone PC10 against proliferating cell nuclear antigen. In in vitro plateau cultures the proportion of PCNA-positive cells exceeded that of Ki-67-positive cells, and only very few cells were negative. In formalin-fixed tissues, the PCNA staining pattern, which should be confined to replicon units in the nucleus, was optimized by 1 h postfixation in an organic solvent (methacarn). Sections showed positive nuclear staining confined to basal and some suprabasal cells in normal urothelium and grade 1 dysplasias, but more generalized nuclear staining in all other neoplastic lesions. In addition, stromal cells adjacent to invasive tumors showed nuclear positivity in some instances. Using quantitative true color image analysis of sections counterstained with hemalum, the degree of brown staining of the PCNA reaction product is contrasted with the blue staining of the nuclear area. With this method low contrast specific staining not appreciated optically can be reliably detected. Image analysis data confirmed observations made on noncounterstained sections and showed significant differences between grade 1 and 2 dysplasias as well as between grade 1 dysplasia and all grades of papillary tumor. Furthermore, a significant difference in PCNA staining indices was found between grade 1 and 3 bladder carcinomas. The results indicate that PCNA staining using the PC10 antibody is not confined to the proliferative fraction of neoplastic urothelium. In contrast with data from normal tissue and malignant hematological neoplasms, the amount of PCNA is regulated differently in urothelial neoplasms, emphasizing the biological differences between the following two sets: mild dysplasia and moderate dysplasia; mild dysplasia and papillary carcinomas. The use of image analysis to standardize the detection process after controlled staining conditions is advisable in order to provide reliable data. Supported by the DFG project: Knuechel/Urothelcarcinom 263     

Cytologic evaluation of low grade transitional cell carcinoma and instrument artifact in bladder washings

OBJECTIVE: To identify key cytologic features for the separation of low grade transitional cell carcinomas (TCCs) from nonneoplastic lesions in bladder washings. STUDY DESIGN: The cytomorphologic features of 95 bladder washing specimens showing papillary fragments, which included 50 low grade TCCs and 45 nonneoplastic lesions, were reviewed retrospectively. RESULTS: Bladder washings from low grade TCCs showed papillary and irregular groups of cells with ragged borders, cytoplasmic homogeneity and subtle nuclear changes, such as increased nuclear/cytoplasmic ratio and irregular nuclear border. Bladder washings after instrumentation from nonneoplastic lesions of the bladder showed cellular specimens with cohesive, ball-shaped and papillary clusters with smooth borders lined with a denser-staining cytoplasmic collar. Reactive urothelial cells often displayed loose aggregates with irregular borders but no cytoplasmic collar. CONCLUSION: In bladder washing cytology, nuclear changes and cytoplasmic homogeneity play a major role in the diagnosis of carcinoma.     

Neural network-based segmentation and classification system for automated grading of histologic sections of bladder carcinoma

OBJECTIVE: To develop an image analysis system for automated nuclear segmentation and classification of histologic bladder sections employing quantitative nuclear features. STUDY DESIGN: Ninety-two cases were classified into three classes by experienced pathologists according to the WHO grading system: 18 cases as grade 1, 45 as grade 2, and 29 as grade 3. Nuclear segmentation was performed by means of an artificial neural network (ANN)-based pixel classification algorithm, and each case was represented by 36 nuclei features. Automated grading of bladder tumor histologic sections was performed by an ANN classifier implemented in a two-stage hierarchic tree. RESULTS: On average, 95% of the nuclei were correctly detected. At the first stage of the hierarchic tree, classifier performance in discriminating between cases of grade 1 and 2 and cases of grade 3 was 89%. At the second stage, 79% of grade 1 cases were correctly distinguished from grade 2 cases. CONCLUSION: The proposed image analysis system provides the means to reduce subjectivity in grading bladder tumors and may contribute to more accurate diagnosis and prognosis since it relies on nuclear features, the value of which has been confirmed.     

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

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

Prognostic value of morphometry in low grade papillary urothelial bladder neoplasms

OBJECTIVE: To analyze the prognostic value of morphometry in low grade papillary urothelial bladder neoplasms (LGPUBNs). STUDY DESIGN: The primary (most common) and secondary (second most common) histologic grades were considered in accordance with the 1998 World Health Organization/International Society of Urological Pathology and the 1999 World Health Organization classifications. With the primary grade, 54 cases were papillary urothelial neoplasms of low malignant potential (PUNLMPs) and 66 low grade papillary urothelial carcinomas (LGPUCs), whereas the secondary grade consisted of 45 PUNLMPs and 75 LGPUCs. To assess the proliferative index, an immunohistochemical study was performed. Regarding nuclear morphometry, an image analysis system on Feulgen-stained sections was utilized in different tumor zones (Zs): Z 1, 100-150 cells from the outer layers of the papillae; Z 2, 100-150 cells from the inner layers; and Z 3, 10 largest nuclei. In univariate studies, a t test, and Mann-Whitney U test and Kaplan-Meier curves were applied, whereas a Cox regression model was used for multivariate study of the variables: size, multiplicity, maximum Ki-67 index, mean nuclear area (MNA) and SD, mean nuclear perimeter and SD, and roundness factor. RESULTS: All 120 cases were followed for a mean of 76.6 months (range, 36-168). In univariate studies, many variables showed a significant correlation (p < 0.05) with recurrence prediction, relapse-free interval and histologic grade regardless of adjuvant therapy. Otherwise, only the MNA of the 10 largest nuclei (threshold, 52 microm2) and the maximum proliferative index (threshold, 7.9%) appeared as independent prognostic markers in the multivariate study. CONCLUSION: In LGPUBNs, the independent prognostic value of MNA of the 10 largest nuclei as well as the maximum proliferative index indicates the importance of histologic grade assessment based on the secondary (second most common) grade.     

An Estimation Procedure for the Joint Distribution of Spatial Direction and Thickness of Flat Bodies Using Vertical Sections Part I: Theoretical Considerations

In soil micromorphology fissures are considered in vertical sections. To get information about the properties of the soil the joint distribution of spatial direction and width is of interest. The fissures are mathematically generalized to flat bodies which form a stationary weighted surface process with the weight “thickness”. Because of stationarity a joint distribution of spatial direction and thickness exists in a “typical point” of the surface process. A suitable parametric family of distributions is assumed. The corresponding parameters can be estimated from measurements on the vertical sections. But on the sections only the visible thickness and the visible angle of a fissure can be measured. Therefore the joint distribution of these variables is expressed by the joint spatial distribution of spatial direction and thickness. This derived distribution depends on the same parameters. The Chi-Square method is proposed for the parameter estimation. The estimation procedure is demonstrated using the Bingham-Mardia distribution for the direction and the lognormal distribution for the thickness and by defining a way to correlate the mean thickness and the direction.     

An Estimation Procedure for the Joint Distribution of Spatial Direction and Thickness of Flat Bodies Using Vertical Sections. Part II: An application in soil micromorphology

In soil micromorphology fissures are considered in vertical sections. To get information about the properties of the soil the joint distribution of spatial direction and width of these fissures is of interest. The fissures are mathematically generalized to flat bodies which are defined as stationary weighted surface processes with the weight “thickness”. In a typical point of the surface process suitable, joint parametric distributions of direction and thickness are assumed. The parameters have to be estimated from measurements on vertical sections which are taken from the soil. On these sections only a visible thickness and a visible angle can be observed. The joint distribution of these variables can be expressed by the joint distribution of spatial direction and thickness with the same parameters and in this indirect way the parameters can be estimated. The paper describes how to randomize the vertical section and how to measure the visible variables on the sections. The Chi-Square method is proposed for the parameter estimation. Further it is discussed how to derive good starting values for the numerical procedure. All this is demonstrated in a simulation study using the Bingham-Mardia distribution for the direction and the lognormal distribution for the thickness including a way to correlate the mean thickness and the direction. Finally an application in soil micromorphology is demonstrated for one soil horizon.